Method for treating dermatitis and improving skin texture using natriuretic peptides

ABSTRACT

The problem to be solved by the present invention is to provide a safe and efficacious therapeutic agent for dermatitis which is not only safe and efficacious for patients with dermatitis, in particular atopic dermatitis, but also significantly effective for severe cases that are judged to be intractable by conventional external preparations, and which is also safely applicable to affected areas such as the face and neck, as well as to subjects with sensitive skin, such as infants and females. Furthermore, the invention aims to provide an external preparation that is efficacious as skin care cosmetics having effects to improve elasticity and wrinkles of the skin, moisturizing effects, and hair-growth effects. The means for solving the problem is a skin external-preparation composition, in particular, a therapeutic agent for dermatitis or a skin texture-improving agent, having C-type natriuretic peptide (CNP) or B-type natriuretic peptide (BNP) as the active ingredient.

This application is a Divisional of Application No. 13/386,528, (nowabandoned), which is a National Phase of PCT International ApplicationNo. PCT/JP2010/062459 filed on Jul 23, 2010, and which claims priorityto patent application Nos. 2009-172589, filed in Japan on Jul 23, 2009and 2010-134600, filed in Japan on Jun 11, 2010. The entire contents ofall of the above applications are hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to a skin external-preparation compositioncomprising C-type natriuretic peptide (CNP) or B-type natriureticpeptide (BNP) as its active ingredient. In particular, the presentinvention relates to a therapeutic agent for skin disease or a skintexture-improving agent comprising C-type natriuretic peptide (CNP) orB-type natriuretic peptide (BNP) as its active ingredient.

BACKGROUND ART

1. Dermatitis:

Dermatitis is an inflammatory reaction of the skin, and is the mostprevalent skin disease. Often in acute skin inflammations, clinically,edematous erythema is presented initially, followed by erythematouslesions with papules and serous papules, then formation of vesicles,pustules, erosions, crusts and scales, followed by healing process. Whena skin inflammation becomes chronic, thickened skin, lichenification andpigmentations are observed, and they are often associated with itching.

Dermatitis includes contact dermatitis, atopic dermatitis, seborrheicdermatitis, nummular eczema, psoriasis, stasis dermatitis, dyshidroticeczema, asteatotic eczema, autosensitization eczema, etc.

Of these, atopic dermatitis is induced by hypersensitive reaction to aforeign protein antigen, a substance derived from other organisms whichexists as house dust in the environment, as well as the involvement ofvarious other non-specific stimulatory responses and specific allergicreactions. It is characterized by pruritic eczema over wide areas of thebody accompanied by dryness and abnormal barrier function of the skin,and many patients have atopic diathesis. Atopic dermatitis is anintractable, chronic inflammatory disease, which relapses betweenremission and exacerbation. It has been known that delayed reactionsassociated with infiltration of eosinophils and lymphocytes, andproduction of various cytokines at the site of inflammation are involvedin the onset and chronicity of atopic dermatitis.

2. Treatment of Dermatitis:

Current treatments for atopic dermatitis involve elimination ofonset/exacerbation factors and skin care, in combination withpharmacotherapy appropriate for symptoms, while steroid externalpreparations are most commonly used for treating dermatitis. Recently,tacrolimus, an immunosuppressive drug, has also been used for treatingatopic dermatitis.

Despite their dramatic clinical effectiveness, however, steroid externalpreparations are drugs that elicit numerous side effects. Steroidexternal preparations are not always satisfactory due to their sideeffects, including skin thinning, atrophy, so-called “moon face” whichresults from fat deposition in the face, skin flush, hirsutism, and skinstriae, etc. In particular, since affected areas in the face and neckhave higher absorbability of drugs than other areas, when a steroidexternal preparation is applied to the face, etc., they are moresusceptible to steroid dermatitis such as steroid-induced rosacea.Moreover, further application of steroid external preparations tocontrol the redness of the skin in steroid dermatitis means that thepatients suffer from a vicious cycle of continuous steroid use. Thisexplains why many patients avoid application of steroid drugs to theface and neck. In addition to these adverse side effects and concernswith regard to the use of steroid external preparations, there arefurther complications with the use of steroid drugs on patients withfairly sensitive skin, including infants, children, women and thepatients with concomitant diseases, with whom eczema and dermatitis arefound frequently. In addition, the repeated and long-term use of steroidexternal preparations, which is necessary due to the chronic nature ofinflammation in atopic dermatitis developed with a background of atopicdiathesis, may result in steroid resistance, in which the steroidexternal preparations become less effective against dermatitis.Furthermore, patients who withdraw from long-term use of steroidexternal preparations often suffer from what is known as a “reboundphenomenon” characterized by aggressive recurrence of a worsenedsymptoms than prior to the treatment.

Other concerns with long-term use of steroid external preparations areskin flush and desquamation of the skin over the whole body, which oftenoccur with the comorbidity of hair loss and swollen lymph nodes. In suchconditions of erythroderma posteczematosa, the patients′ social livesare severely disrupted.

Generally, steroids used for treating atopic dermatitis are classifiedas the “strongest”, “very strong”, “strong”, “medium” and “weak” inorder of their efficacy, and the choice depends on factors such as thebody region, severity of the rash, age and the period of use. For atopicdermatitis treatment using steroids, it is recommended that a steroid ofa rank that is sufficiently strong to suppress the inflammatory symptomsof atopic dermatitis is prescribed initially, which is then replaced byprogressively weaker ranks of steroids once the symptoms are controlled,allowing the earliest withdrawal from the steroid use.

The “strongest” rank steroids include: clobetasol propionate anddiflorasone diacetate, and “very strong” steroids include mometasonefuroate, betamethasone butyrate propionate, fluocinonide, betamethasonedipropionate, difluprednate, budesonide, amcinonide, diflucortolonevalerate, and hydrocortisone butyrate propionate. In addition, the“strong” rank steroids includes: deprodone propionate, dexamethasonepropionate, dexamethasone valerate, halcinonide, betamethasone valerate,beclomethasone propionate, fluocinolone acetonide, etc.; and “medium”rank steroids include: prednisolone valerate acetate, triamcinoloneacetonide, flumethasone pivalate, alclometasone propionate, clobetasonebutyrate, and hydrocortisone butyrate. The “weak” rank steroids include:predonisolone and hydrocortisone acetate.

Patients with chronic atopic dermatitis due to repeated recurrence areoften found to have a history of use of multiple steroids belonging toeither the “strongest” or “very strong” rank.

On the other hand, the alternative external preparation available, i.e.,tacrolimus, is an immunosuppressive agent, and hence it is notapplicable to children with 6 years old of age or younger, women who areor may be pregnant, and patients with renal disorders. The applicationof tacrolimus also significantly increases the risk of complication ofherpes virus infection. This is thought to be due to the effect of thedisturbance in the barrier function of the skin suffering from dryness,combined with the steroid-induced depression in the local immunefunctions. More seriously, incidence of malignant tumors has beenrecently reported in infants and children, making the safety oftacrolimus questionable. In addition, some patients find some of thelocal strong irritative side effects including burning sensation anditching, which inevitably occur upon external application of tacrolimus,unbearable.

For the reasons above, a safe, effective, adverse effect-freetherapeutic agent for dermatitis, which is not only applicable withoutirritation to affected areas of higher absorbability of drugs with highdensity of hair follicles such as the face and the neck, but also safeto apply to patients having sensitive skins, such as females andchildren, is much hoped for.

3. Natriuretic Peptides:

Natriuretic peptides (NPs) are classified into three known families,named atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP),and C-type natriuretic peptide (CNP); their well-known members arecomposed of 28, 32, and 22 amino acid residues, respectively.

(1) ANP and BNP:

ANP and BNP are synthesized mainly by the atria and the ventricles,respectively, and released from the heart into the whole body. It isthought that nearly 100% of the circulating ANP and BNP in the bloodoriginate from the heart. These ANP and BNP are reported to be deeplyinvolved in numerous diseases, including hypertension, cardiomegaly,cardiac failure, myocardial infarction, valvular heart disease, cardiacdysrhythmia, and pulmonary hypertension.

Human ANP is a peptide produced and released by atrial cardiocytes, andis composed of 28 amino acids, of which the 7^(th) cysteine and the23^(rd) cysteine are bonded by a disulfide bond to form a ringstructure. ANP has been shown to have diuretic effects in the kidneysand relaxes/dilates vascular smooth muscle cells in the blood vessels.In contrast, human BNP is a peptide produced and released by ventricularcells, and is composed of 32 amino acids, of which the 10^(th) cysteineand the 26^(th) cysteine are bonded by a disulfide bond to form a ringstructure. BNP also possesses both diuretic and vasodilatory effects.BNP was originally isolated and identified in the porcine brain in Japanin 1988, and is also called B-type natriuretic peptide.

Both ANP and BNP bind to the receptor NPR-A (also called GC-A) having aguanylate cyclase domain, and exert their effects stated above, bystimulating the production of cGMP. In fact, secretion of ANP isstimulated in response to an increase in the atrial pressure by itsdistension in congestive heart failure, etc., and through its actionstated above, ANP relieves the symptoms of congestive heart failure,etc. Likewise, BNP's release is stimulated during certain conditionsincluding myocardial infarction, and BNP, through its action mentionedabove, relieves the symptoms associated with myocardial infarction, etc.(Refer to non-patent literature 1). Although most of the circulating BNPderives from the ventricles, some BNP is released by the atria. Incardiac failure, the level of expression of both ANP and BNP increasesto as much as 100 times more the normal level, but the increase of BNPexpression is reported to be both greater and faster than that of ANP.While ANP (hANP) is marketed as a prescription drug for treating acutecardiac failure in Japan, BNP is clinically used in the United States.

(2) CNP:

CNP, which was once thought to function only as a brain peptide becauseit was first found in the brain, has now been clarified to exist in theperiphery as well. In the vessel walls, in particular, CNP specificreceptors were found to be abundant in smooth muscle cells, and CNP isproduced by cells of the monocyte/macrophage lineage and the endothelialcells. For those reasons, CNP is speculated to function in the vascularwalls as a local mediator involved in inhibition of growth of vascularsmooth muscle cells. Its clinical application is currently beinginvestigated for possible prevention of restenosis by CNPadministration, which occurs with a certain frequency after percutaneoustransluminal coronary angioplasty (PTCA) performed on patients withischemic heart failure.

Recently it has been reported that intravenous administration of CNPremarkably improves cardiomegaly and fibrosis associated with myocardialinfarction, and improves cardiac functions in animal experiments.Cardiac fibrosis is known to cause diastolic ventricular failure andcardiac dysrhythmia. Since CNP possesses a powerful action to suppressfibroblast proliferation, the potential of CNP as an anti-fibroticmedication for the heart is under investigation. Since CNP is a hormonenaturally occurring in the body, there is only little concern of ithaving adverse side effects; accordingly clinical application of CNP asa therapeutic agent for arteriosclerotic diseases and heart diseases isexpected. Here, examples of CNP include CNP-22 composed of 22 aminoacids, and CNP-53 wherein 31 amino acid residues are attached to theN-terminal of CNP-22.

(3) Natriuretic Peptide Receptors:

Natriuretic peptide receptors are classified into three subtypes; NPR-Areceptor (also called GC-A) and NPR-B receptor (also called GC-B) bothof which contain a guanylate cyclase domain, and NPR-C receptor whichlacks a guanylate cyclase domain. It is known that ANP can bind to NPR-Aand NPR-C receptors, BNP can bind to NPR-A and NPR-C receptors, and CNPcan bind to NPR-B and NPR-C receptors.

It is suggested that the activation of NPR-A receptors inducesvasodilatory action, diuretic action, and cell growth inhibitory action,while NPR-B receptors are abundant in vascular smooth muscle cells andthought to be involved in the growth inhibition of vascular smoothmuscle cells.

(4) Relationship Between Natriuretic Peptides and Immune System:

Historically, natriuretic peptide was first discovered as a peptidereleased from the atria, later named ANP, and its vasodilatory anddiuretic actions gathered attention. BNP and CNP were then discovered aspeptides similar to ANP. This historical background offers anexplanation to why any attention to the relationship between natriureticpeptides and the immune system has been focused on those related to thecardiovascular system. CNP knock-out mice demonstrated impaired growthof cartilage resulting in a dwarfism-like phenotype (refer to Non-patentliterature 2), which directed some interest to the relationship betweenarthritis and natriuretic peptides.

ANP is implicated in playing a role in arthritis and sepsis as itinhibits the release of inflammatory cytokines including tumor necrosisfactor (TNF-α) and interleukin 1β (IL-1β) by macrophages (refer toNon-patent literature 3). This literature, however, does not mentionANP's relationship with the skin.

Similarly, the blood concentration of BNP has been reported to increasewith the rejection response following heart transplant, and therefore itis suggested that it is associated with the immune regulation in thecardiovascular system (refer to Non-patent literature 4). However, thisliterature does not describe any connection between BNP and the skin.

Taking into account the observation that there is an increase in theblood concentration of BNP during the heart graft rejection, Kuroski deBold et al. have investigated the immunoregulatory action of natriureticpeptides, and have demonstrated that both ANP and BNP inhibit thelymphocyte growth (refer to Non-patent literature 5). However, there isno connection between natriuretic peptides and the skin mentioned inthis literature.

Chiurchiu et al. on the other hand have investigated theimmunoregulatory action of BNP focusing on its association with theheart diseases and sepsis, and showed that BNP promotes the release bymacrophages of pro-inflammatory cytokines such as arachidonic acid,prostaglandin E2 (PGE2), and leukotriene B4 (LTB4), and also promotesthe release of anti-inflammatory cytokines including interleukin 10(IL10). Thus, while BNP is indicated to have some action in theregulation of inflammatory responses, whether BNP acts overall tosuppress or promote inflammatory responses remains inconclusive in theliterature (refer to Non-patent literature 6). This literature also doesnot mention any connection between BNP and the skin.

Similarly, CNP is reported to be released by macrophages (refer toNon-patent literature 7), and while investigating the roles of CNP incardiac ischemia and myocardial damage after reperfusion, Scotland etal. report that CNP suppresses platelet aggregation and lymphocytemigration (refer to Non-patent literature 8). The connection between CNPand the skin, however, is not described in this literature.

Likewise, Obata et al. examined the roles played by CNP in myocarditisusing a rat myocarditis model generated by injecting pig myosin. Theyreported that continuous administration of CNP for 1 week had suppressednecrosis and inflammation of the cardiac tissues, while at the same timepromoted the regeneration of blood vessels, thereby preventingfunctional loss of the heart (refer to Non-patent literature 9).Nevertheless, there is nothing in this literature to suggest aconnection between CNP and the skin.

In addition, based on the observation that CNP knock-out mice show adwarfism-like phenotype, attention has been paid to the potentialconnection between CNP and cartilage growth. Agoston et al. demonstratedthat when incubated with Dexamethasone, the primary culturedchondrocytes extracted from the tibial bones of mouse embryos hadsignificantly increased the expression of CNP genes (refer to Non-patentliterature 10). This literature, however does not describe anyconnections between CNP and the skin.

It is evident that the connections between natriuretic peptides and theimmune system have drawn increasing attention in recent years, but it islimited only to the inflammation of the cardiovascular system andarthritis, and the relationship between dermatitis or more specificallyatopic dermatitis and natriuretic peptides has never been reported.

(5) Reports on the Application of Natriuretic Peptides:

Following are some examples of a number of applications of CNP, BNP andANP.

Toshiko Koide and her colleagues have proposed a preparation forrepair/regeneration of tissues and organs, comprising a composition thatcomprises ANP, BNP, CNP and urodilatin (P-Uro), and their precursors andderivatives, or combinations thereof as an active ingredient, and thatmay comprise pharmaceutically commonly-used diluents, excipients,fillers, and auxiliary agents (refer to Patent Literature 1).

However, specific examples of repair and regeneration of tissues andorgans relate only to the regeneration of myocardiocytes, hypodermaltissue, hair, and improvement of cracked, rough skin due to wet work;they all correspond to ANP administration. There is no statement whichimplies therapeutic agents for treating skin disease or skintexture-improving agents by means of administration of CNP or BNP.

Masaharu Tanaka and his colleagues have proposed a C-type natriureticpeptide exhibiting a growth inhibitory action of vascular smooth musclecells, as well as a growth inhibitory preparation for vascular smoothmuscle cells containing such peptides as its active ingredient (refer toPatent Literature 2).

This, however, relates to the use of CNP in a growth inhibitory agent ofvascular smooth muscle cells but does not imply application of CNP orBNP to therapeutic agents for dermatitis.

Katsuhiko Nakada and his colleagues proposed an eye drop for promotinglacrimal secretion or for treating keratoconjunctival disorder,containing as its active ingredient a natriuretic peptide, and they listANP, BNP and CNP as examples of usable natriuretic peptide (refer toPatent Literature 3).

This, however, only relates to the application of the property of ANP,CNP and BNP to promote lacrimal secretion in an eye drop for treatingkeratoconjunctival disorder, and does not indicate the use of CNP or BNPin a therapeutic agent to treat dermatitis.

Kazuwa Nakao and his colleagues proposed a composition for increasingthe body length containing a guanyl cyclase B (GC-B) activator as theactive ingredient, which is to be administered to an individual withoutFGFR3 abnormality (refer to Patent Literature 4).

This indicates an application of CNP in a composition for increasing thebody height based on the finding that the nose-anus length in transgenicmice which over-expressed CNP was larger than that in normal litters,but dose not imply the use of CNP or BNP in a therapeutic agent fordermatitis.

Kazuwa Nakao and his colleagues also proposed a prophylactic agent ortherapeutic agent for inflammation of the joints containing a guanylcyclase B (GC-B) activator such as CNP as an active ingredient (refer toPatent Literature 5).

However, this relates only to the application of CNP in a therapeuticagent or prophylactic agent for inflammation of the joints based on astudy revealing that, compared to their litter mates, the articularcartilages grow thicker in the transgenic mice which over-express CNP,along with the observation that arthritis is repressed by the continuousadministration of CNP to model animals of arthritis. Hence this does notimply the application of CNP or BNP in a therapeutic agent fordermatitis.

In addition, Masaharu Tanaka and his colleagues reported that CNPdiffers from ANP and BNP in the structure and function effects as statedbelow (refer to Patent Literature 2).

“At present, both ANP and BNP are thought to act as a hormone secretedby the heart into the blood, as well as a neurotransmission factor, andto play an important role in maintaining the amount of body fluid andhomeostasis of blood pressure . . . . There are many unknown points inthe physiological roles of CNP as a natriuretic peptide. Namely, sinceCNP has an amino acid primary sequence similar to that of ANP and BNPand shows a natriuretic action and a hypotensive action by in vivoadministration, CNP was relegated to the natriuretic peptide family.However, because the natriuretic action and hypotensive action of CNPare significantly weaker than those of ANP and BNP (from 1/50 to 1/100). . . , CNP has held a unique position in the natriuretic peptidefamily, and has been presumed to be playing a role different from themaintenance of amounts of body fluid and homeostasis of blood pressure .. . . Comparing the structure of CNP with that of ANP/BNP, CNP differsfrom ANP or BNP in the following points. Namely, the primary amino acidsequence of CNP differs from that of ANP or BNP at the exocyclicN-terminal domain; of the 17 amino acid residues in the endocyclicdomain, 5 residues and 4 residues in CNP differ from those in ANP andBNP, respectively. In addition, the structure of the exocyclicC-terminal domain of CNP largely differs from that of ANP or BNP, andCNP does not have the tail structure which exists in ANP or BNP (in thecases of ANP and BNP, 5 amino acid residues and 6 amino acid resides,respectively, are attached to the C-terminal of the cyclic structure inANP and BNP; this structure is called a tail structure for descriptivepurposes). Thus-described structural differences between CNP and ANP/BNPare obviously involved in the manifestation of the above-mentionedcharacteristic pharmacological effects of CNP.”

REFERENCE LIST Patent Literature

-   Patent Literature 1: JP A 2008-162987-   Patent Literature 2: JP A 6-9688-   Patent Literature 3: JP A 2000-169387-   Patent Literature 4: WO 2005/094890-   Patent Literature 5: WO 2005/094889

Non-Patent Literature

-   Non-patent Literature 1: European J. Endocrinology, Vol. 135, p.    265, 1996.-   Non-patent Literature 2: Proceedings of the National Academy of    Sciences of the United States of America, Vol. 98, No. 7, p. 4016,    2001.-   Non-patent Literature 3: Annals of the Rheumatic Disease, Vol. 60,    Suppl 3, iii, p. 68, 2001.-   Non-patent Literature 4: The Journal of Heart and Lung    Transplantation, Vol. 27, p. 31, 2008.-   Non-patent Literature 5: The Journal of Heart and Lung    Transplantation, Vol. 29, No. 3, p. 323, 2010.-   Non-patent Literature 6: Regulatory Peptides, Vol. 148, p 26, 2008.-   Non-patent Literature 7: Experimental Hematology, Vol. 29, p. 609,    2001.-   Non-patent Literature 8: Proceedings of the National Academy of    Sciences, Vol. 102, No. 40, p. 14452, 2005.-   Non-patent Literature 9: Biochemical and Biophysical Research    Communications, Vol. 356, p. 60, 2007.-   Non-patent Literature 10: BMC Musculoskeletal Disorders, Vol. 7, p.    87, 2006.

DISCLOSURE OF INVENTION Summary of Invention Problems to be Solved bythe Invention

The chronic nature of dermatitis, a representative skin disease, and inparticular atopic dermatitis, makes continuous use of drugs necessary.While steroid external preparations produce satisfactory clinicaleffects as stated above, they also have many adverse local effects,including skin thinning and atrophy, moon face, skin flush, hirsutism,and skin striae, hence their application to areas of high drugabsorbability such as the face, and to patients with sensitive skin,children and females, has been difficult. In addition to the problem ofadverse local effects, the repeated and long-term use of topicalsteroids, which is necessary in many cases due to the chronic nature ofatopic dermatitis, can lead to rebound phenomena induced bydiscontinuation of external application. In other words, an acuteexacerbation, which is worse than the state before the application,occurs upon abrupt discontinuation of external application. In addition,as more serious complications, erythroderma posteczematosa withwhole-body skin flush and desquamation can result from continuousinappropriate external application. These severe symptoms induced by along-term use of steroids have been a serious problem.

Hence, the aim of the present invention is to provide an effective andsafe skin external preparation composition for patients with dermatitis,atopic dermatitis in particular, which is not only effective againstsevere conditions which are intractable with the currently availableexternal preparations, but also safely applicable to affected areas ofthe face and neck, and also safely applicable to patients with sensitiveskin, children and females. Furthermore, the invention also aims toprovide a skin texture-improving agent to enhance the skin barrierfunctions important for prevention of recurrence, to improve the textureand moisture retention ability of the skin, which has ananti-inflammatory effect and effects of care for the horny cell layer,care for the epidermis, and care for the basal membrane.

Means for Solving Problems

Through extensive research, the inventors of the present inventiondiscovered that C-type natriuretic peptide (CNP) and B-type natriureticpeptide (BNP) are safe and effective as a therapeutic agent fordermatitis, especially atopic dermatitis, and that they are alsoapplicable to the face and neck, and patients who have sensitive skins,children and females. The present invention was thus made.

The present invention comprises the following.

-   1. A skin external-preparation composition comprising C-type    natriuretic peptide (CNP) or B-type natriuretic peptide (BNP).-   2. The skin external-preparation composition according to the item    1, wherein the C-type natriuretic peptide (CNP) or B-type    natriuretic peptide (BNP) is a chimeric peptide of CNP and BNP, in    which CNP is CNP-22, CNP-53, or a peptide comprising any amino acid    sequence with 5 or more consecutive amino acids in the amino acid    sequence having deletion, substitution, or addition of any amino    acid in the amino acid sequence of CNP-22 or CNP-53, and in which    BNP is BNP-26, BNP-32, BNP-45, or a peptide comprising any amino    acid sequence with 5 or more consecutive amino acids in the amino    acid sequence having deletion, substitution, or addition of any    amino acid in the amino acid sequence of BNP-26, BNP-32 or BNP-45,    and wherein the chimeric peptide forms a ring structure by an    intramolecular disulfide bond, and wherein the chimeric peptide has    CNP activity or BNP activity; or derivative(s) of the chimeric    peptide.-   3. The skin external-preparation composition according to the item    1, wherein the C-type natriuretic peptide (CNP) is CNP-22, CNP-53,    or a CNP derivative in which any amino acid in the amino acid    sequence of CNP-22 or CNP-53 is deleted, substituted or added, and    which has CNP activity.-   4. The skin external-preparation composition according to the item    3, wherein the C-type natriuretic peptide (CNP) is CNP-22.-   5. The skin external-preparation composition according to the item    1, wherein the B-type natriuretic peptide (BNP) is BNP-26, BNP-32,    BNP-45, or a BNP derivative in which any amino acid in the amino    acid sequence of BNP-26, BNP-32, or BNP-45 is deleted, substituted    or added, and which has BNP activity.-   6. The skin external-preparation composition according to the item    5, wherein the B-type natriuretic peptide (BNP) is BNP-32.-   7. The skin external-preparation composition according to the item    1, comprising 1-500 μg/g of C-type natriuretic peptide (CNP) or    B-type natriuretic peptide (BNP).-   8. The skin external-preparation composition according to the item    1, comprising 20-200 μg/g of C-type natriuretic peptide (CNP) or    B-type natriuretic peptide (BNP).-   9. The skin external-preparation composition according to the item    1, comprising 30-100 μg/g of C-type natriuretic peptide (CNP) or    B-type natriuretic peptide (BNP).-   10. The skin external-preparation composition according to the item    1, wherein the skin external-preparation composition is a    therapeutic agent for dermatitis or a skin texture-improving agent.-   11. The skin external-preparation composition according to the item    10, wherein the skin external-preparation composition is a    therapeutic agent for dermatitis, and the dermatitis is atopic    dermatitis, dermatitis that led up to steroid dermatitis,    steroid-resistant dermatitis, dermatitis to which tacrolimus is not    applicable, chronic dermatitis, erythroderma, eczema, contact    dermatitis, seborrheic dermatitis, autosensitization dermatitis,    stasis dermatitis, urticaria, drug eruption, dermal vasculitis,    prurigo, pruritus cutaneus, erythema, psoriasis, rosacea,    rosacea-like dermatitis, lichen planus, or follicular keratosis.-   12. The skin external-preparation composition according to the item    11, wherein the dermatitis is atopic dermatitis-   13. The skin external-preparation composition according to the item    11, wherein the dermatitis is dermatitis that led up to steroid    dermatitis.-   14. The skin external-preparation composition according to the item    11, wherein the dermatitis is steroid-resistant dermatitis.-   15. The skin external-preparation composition according to the item    11, wherein the dermatitis is the one for which tacrolimus is not    applicable.-   16. The skin external-preparation composition according to the item    11, wherein the dermatitis is chronic dermatitis.-   17. The skin external-preparation composition according to the item    11, wherein the dermatitis is eczema.-   18. The skin external-preparation composition according to the item    11, wherein the dermatitis is erythroderma.-   19. The skin external-preparation composition according to the item    11, wherein the dermatitis is rosacea.-   20. The skin external-preparation composition according to the item    11, wherein the dermatitis is rosacea-like dermatitis.-   21. The skin external-preparation composition according to the item    11, wherein the dermatitis is psoriasis.-   22. The skin external-preparation composition according to the item    10, wherein the skin external-preparation composition is a    therapeutic agent for dermatitis, and the dermatitis is an    inflammation associated with at least one rash symptom selected from    erythema, infiltrative erythema, lichenified lesion, scales,    adhesion of crusts, eczema, abrasion, excoriation, prurigo    nodularis, papule, erosion, infiltration, vesicle, and edema.-   23. The skin external-preparation composition according to the item    10, wherein the skin external-preparation composition is a    therapeutic agent for dermatitis, and the dermatitis shows an immune    reaction to at least one allergen selected from house dust, mite,    cedar pollen, orchard grass pollen, ragweed pollen, egg white, and    egg yolk.-   24. The skin external-preparation composition according to the item    10, wherein the skin external-preparation composition is a    therapeutic agent for dermatitis, and the dermatitis occurs in at    least one region selected from the face, neck, back, and arms.-   25. The skin external-preparation composition according to the item    1, wherein the dosage form of the external preparation is selected    from ointment, gel, cream, lotion, solution, spray, and patch.-   26. The skin external-preparation composition according to the item    25, wherein the dosage form is ointment, gel, cream, or solution.-   27. The skin external-preparation composition according to the item    1, wherein the skin external-preparation composition is a skin    texture-improving agent.-   28. The skin external-preparation composition according to the item    27, wherein the skin external-preparation composition is a skin    texture-improving agent for improving dry skin, rough skin,    sensitive skin and fine wrinkles.-   29. The skin external-preparation composition according to the item    27, wherein the skin texture-improving agent is a skin care product    or a quasi drug.-   30. The skin external-preparation composition according to the item    27, wherein the dosage form is cream, foam, skin lotion, facial    mask, skin-softening water, skin emulsion, foundation, makeup base,    essence, soap, liquid cleanser, bath agent, sun-block cream, suntan    oil or spray-type liquid preparation.

Effects of the Invention

As described in the examples given below, a skin external-preparationcomposition of the present invention comprising CNP or BNP as its activeingredient can greatly improve erythema, papules and scales associatedwith severe swelling/edema/infiltration or lichenification, therebyachieving either remission, mild conditions mainly characterized by dryskin with mild erythema and scales, or minor rash having littleinflammatory symptoms with dryness of the skin as its main symptom.Hence, the therapeutic agent for dermatitis of the present invention isexpected to be applicable and extremely effective against atopicdermatitis, which develops resistance to the conventional steroidexternal drugs. In particular, it is possible to dramatically improveerythema, infiltration, scales or lichenification and burning sensationon the adult face without producing irritative symptom, which interferewith many aspects of social life. Remarkable improvements are also madein the other parts of the body including the upper limbs and back. Theeffects are not limited to adults but are similar with infants.

Conventionally and widely used steroid external preparations have thedrawbacks of, upon discontinuation of the external application,relapsing to the severity of pre-treatment conditions, or worse, therebound phenomena mainly characterized by the recurrence of worsenedcondition. In contrast, the present invention not only completely lacksthese problems, but also results in moisturized, fine-textured skin. Theeffects of the therapeutic agent for dermatitis of the present inventionlasted 5 days to 2 weeks after its application was terminated, and theimproved skin conditions were sustained throughout. Even in cases, whichdid not result in the prominent effect, the therapeutic goals of atopicdermatitis were achieved, namely, the main symptoms were improved todryness, minor or mild severity of erythema, and scales, etc.Furthermore, minor erythema, edema, papules and scales, which recurredafter the treatment in some cases, did not worsen to the pre-treatmentconditions and remained stable. This could not be attained by thesteroid external preparations, the former therapeutic option, and istherefore worthy of special mention. In addition, when re-applied to therashes that had relapsed, CNP or BNP preparations of the presentinvention required a smaller number of applications than that of theinitial time to reduce the severity of symptoms to either mild or minorrash. In terms of manifestation of effects, the patients experiencedsubjective awareness of burning sensation subsiding approximately 10 minafter the application, and this was followed by the improvements ofobservable symptoms including erythema, infiltration, edema, papules,scales and excoriations after approximately 30 min. When application wasfurther continued from 2 days to 4 days, erythema and infiltrationimproved remarkably and the skin became near-normal in condition with afine texture in many cases. These findings were astonishing even for theinventor of the present invention who is a dermatologist. CNPpreparations demonstrated especially remarkable effects.

Given that both BNP and ANP belong to the same family and share the samereceptors, it was formerly assumed that BNP and ANP preparationspossessed equivalent effects. When they were actually tested on patientswith inflammatory disease, in particular atopic dermatitis, however, BNPpreparations were revealed to have much more intense pharmacologicaleffects than ANP preparations. That is to say that BNP preparations arefaster-acting than ANP preparations, and lead to better improvements ofthe clinical symptoms and the effects lasted longer. On the other hand,ANP preparations unexpectedly resulted in much poorer improvements inthe local dermal symptoms of erythema, infiltration, scale andlichenification compared to BNP preparations, and in many cases, thesymptoms showed no improvements or worsened. In cases where a littleimprovement was observed with ANP preparations, manifestation of theeffects was slow and even 7 consecutive days of external application didnot result in complete remission of erythema, while both erythema anddryness of the skin remained in all cases. The finding that BNP as askin external-preparation composition had more intense pharmacologicaleffects than ANP belonging to the same family of natriuretic peptide wassurprising.

The active ingredients of the present invention, CNP and BNP, arehormones which naturally occur in the body. Side effects are lessexpected and with adequate dosage, they are thought to have only a minoreffect on the hemodynamic status and hence they are safe to apply topatients with low or unstable blood pressure, allowing long-termadministration to chronic dermatitis patients. They show a potency todermatitis greater than that of conventional steroid externalpreparations and are also rapid-acting to a greater degree and lead tolonger-lasting effects, or more precisely, 5 days to 2 week remissionperiods were observed even in the severe cases. Moreover, the skinexternal-preparation composition of the present invention is safelyapplicable to patients with sensitive skin, children and females, and tothe face and neck region, etc. without irritation symptoms, and thismakes the present invention an unprecedented, important therapeuticagent for dermatitis.

In addition, when a skin texture-improving agent comprising CNP or BNPof the present invention was applied, effects such as improvement of thetexture, dryness and roughness of the skin while enhancing the softnessand moisture retention, as well as obscuring fine wrinkles wereconfirmed. The present invention can therefore be intended as both atherapeutic agent for dermatitis and a skin texture-improving agentaimed at skincare products or similar for improving elasticity, wrinklesof the skin and moisture retention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph showing the effects of a CNP gel preparation ofthe present invention when it was applied to a patient who had severeswelling, infiltration and erythema on the upper limbs. A and B show thestate before application. C shows the state after application of the CNPgel preparation with 30 μg/g concentration three times at 20 minintervals, while D shows the state after application of a gelpreparation without the addition of CNP 3 times at 20 min intervals.(Refer to Case 10 of the CNP gel preparation; subject 5: Tables 3 and 4)

FIG. 2 is a photograph showing the effects of a CNP aqueous-solutionpreparation of the present invention when it was applied to a patientwho had a rash mainly characterized by severe swelling, infiltration,erythema with lichenification, papules, erosions, and numerousexcoriations on the face. A shows the state before application, and Bshows the state after application of the CNP aqueous-solutionpreparation with a concentration of 100 μg/ml twice a day for 4 days.(Refer to Case 2 of the CNP aqueous-solution preparation; subject 6;Tables 5 and 6)

FIG. 3 is a photograph showing the effects of a CNP aqueous-solutionpreparation of the present invention when it was applied to a patientwho had a rash mainly characterized by erythema with lichenification,papules, erosions, scales and numerous excoriations on the face. A showsthe state before application, and B shows the state after application ofthe CNP aqueous-solution preparation with a concentration of 100 μg/mltwice a day for 4 days. (Refer to Case 7 of CNP aqueous-solutionpreparation; subject 7; Tables 5 and 6)

FIG. 4 is a photograph showing the effects of a CNP gel-base preparationof the present invention when it was applied to the arm of a patient whopresented with erythema associated with lichenification, infiltrativeerythema, severe scales, adhesion of crusts, vesicles, and erosionsthroughout the whole body. A shows the state before application, and Bshows the state after application of the CNP gel-base preparation with aconcentration of 30 μg/g, twice a day for 2 days. (Refer to CNP gel-basepreparation; subject 17; Tables 9 and 10)

FIG. 5 is a photograph showing the effects of a CNP gel-base preparationof the present invention when it was applied to a patient who presentedwith a rash associated with severe swelling/edema/infiltration/erythemaon the upper limbs. A shows the state before application, and B showsthe state after application of the CNP gel-base preparation with aconcentration of 50 μg/g, twice a day for 4 days. (Refer to CNP gel-basepreparation; subject 20; Tables 9 and 10)

FIG. 6 is a photograph showing the effects of a CNP ointment preparationof the present invention, when it was applied to the face and neck of apatient who presented with infiltrative erythema, erythema, severescales, adhesion of crusts, and numerous excoriations over the wholebody, and particularly apparent on the face and neck. A shows the statebefore application, and B shows the state after application of the CNPointment preparation with a concentration of 30 μg/g twice a day for 2days. (Refer to CNP ointment preparation; subject 21; Tables 11 and 12)

FIG. 7 is a photograph showing the effects of a CNP ointment preparationof the present invention when it was applied to the face of a patientwho presented with infiltrative erythema with strong itching associatedwith sleep disturbance, erythema, excoriations on the face, neck, fourlimbs, and back, and had a rash mainly characterized by severeinfiltrative erythema, scales, and numerous excoriations particularly onthe face. A shows the state before application, and B shows the stateafter application of the CNP ointment preparation with a concentrationof 50 μg/g, twice a day for 3 days. (Refer to CNP ointment preparation;subject 23; Tables 11 and 12)

FIG. 8 is a photograph showing the effects of a CNP ointment preparationof the present invention, when it was applied to the neck and back of apatient who presented with infiltrative erythema, lichenified lesions,erythema, adhesion of crusts, and numerous excoriations on the bodytrunk, and had a rash forming a plaque associated with erythema withsevere infiltration/lichenification, severe scales, and adhesion ofcrusts on the back. A shows the state before application, and B showsthe state after application of the CNP ointment preparation with aconcentration of 50 μg/g twice a day for 3 days. (Refer to CNP ointmentpreparation; subject 29; Tables 13 and 14)

FIG. 9 is a photograph showing the effects of a CNP ointment preparationof the present invention, when it was applied to the upper limb of apatient suffering from the condition of erythroderma posteczematosa whopresented infiltrative erythema, lichenified lesions, erythema, severescales, adhesion of crusts, numerous excoriations over the whole body,and who had a rash mainly characterized by erythema associated withsevere swelling and infiltration on the upper limbs. A shows the statebefore application, and B shows the state after application of the CNPointment preparation with a concentration of 50 μg/g, twice a day for 2days. (Refer to CNP ointment preparation; subject 30; Tables 13 and 14)

FIG. 10 is a photograph showing the effects of a CNP ointmentpreparation of the present invention when it was applied to the face ofa patient who presented a rash consisting of infiltration/severe scales,adhesion of crusts, erosions and numerous excoriations on the face, andalso infiltrative erythema, lichenified lesions, erythema, severescales, adhesion of crusts, and numerous excoriations over the wholebody. A shows the state before application, and B shows the state afterapplication of the CNP ointment preparation with a concentration of 50μg/g twice a day for 3 days. (Refer to CNP ointment preparation; subject27; Tables 13 and 14)

FIG. 11 is a photograph showing the effects of a CNP ointmentpreparation of the present invention when it was applied to the face ofa patient suffering from the condition of erythroderma posteczematosawho presented a rash mainly characterized by infiltrative erythema withswelling and erosions on the face and neck, and infiltrative erythema,lichenified lesions, erythema, severe scales, and adhesion of cruststhroughout the whole body. A shows the state before application, and Bshows the state after application of the CNP ointment preparation with aconcentration of 50 μg/g twice a day for 3 days. (Refer to CNP ointmentpreparation; subject 28; Tables 13 and 14)

FIG. 12 is a photograph showing the effects of a BNP gel-basepreparation of the present invention when it was applied to a patientwho had a rash mainly characterized by severe edema, infiltration,erythema, erosions, scales and numerous excoriations on the neck. Ashows the state before application, and B shows the state afterapplication of the BNP gel-base preparation with a concentration of 50μg/g twice a day for 5 days. (Refer to Case 1 of BNP gel-basepreparation; subject 41; Tables 19 and 20)

FIG. 13 is a photograph showing the effects of a BNP gel-basepreparation of the present invention when it was applied to a patientwho had a rash on the forearms, characterized by infiltration, erythema,severe scales and crusts. A shows the state before application, and Bshows the state after application of the BNP gel-base preparation with aconcentration of 50 μg/g twice a day for 5 days. (Refer to Case 4 of BNPgel-base preparation; subject 42; Tables 19 and 20)

FIG. 14 is a photograph showing the effects of a BNP gel-basepreparation of the present invention when it was applied to a patientwho had a rash on the face and neck, mainly characterized by erythemawith edema, erosions, scales and numerous excoriations. A shows thestate before application, and B shows the state after application of theBNP gel-base preparation with a concentration of 50 μg/g twice a day for2 days. (Refer to Case 5 of BNP gel-base preparation; subject 40; Tables17 and 18)

FIG. 15 is a photograph showing the effects of a BNP aqueous-solutionpreparation of the present invention when it was applied to a patientwho had a rash on the face, mainly characterized by infiltrativeerythema, many papules, scales and excoriations. A shows the statebefore application, and B shows the state after application of the BNPaqueous-solution preparation with a concentration of 50 μg/ml twice aday for 5 days. (Refer to Case 8 of BNP aqueous-solution preparation;subject 43; Tables 19 and 20)

FIG. 16 is a photograph showing the effects in a comparative case whenan ANP gel-base preparation was applied to a patient who had a rash onthe face, mainly characterized by severe lichenified infiltration,erythema and scales. A shows the state before application, and B showsthe state after application of the ANP gel-base preparation with aconcentration of 50 μg/g twice a day for days. (Refer to Case 2 of ANPgel-base preparation; subject 46; Tables 21 and 22)

FIG. 17 is a photograph showing the effects in a comparative case whenan ANP gel-base preparation was applied to a patient who had a rash onthe face, neck and the four limbs, characterized by severe swelling,skin flush and edema. A shows the state before application, and B showsthe state after application of the ANP gel-base preparation with aconcentration of 50 μg/g twice a day for days. (Refer to Case 3 of ANPgel-base preparation; subject 45; Tables 21 and 22)

FIG. 18 is a photograph showing the effects in a comparative case whenan ANP gel-base preparation was applied to a patient who had a rash onthe back, characterized by severe infiltration, erythema, numerousexcoriations, papules and lichenification. A shows the state beforeapplication, and B shows the state after application of the ANP gel-basepreparation with a concentration of 50 μg/g twice a day for 5 days.(Refer to Case 5 of ANP gel-base preparation; subject 48; Tables 21 and22)

FIG. 19 is a graph showing the changes in the severity of the rashesevaluated using SCORAD before and after application of the skinexternal-preparation compositions comprising CNP or BNP of the presentinvention. The white bars represent severity levels before application,while the black bars represent severity levels after application. Thelength of the bars represents the average, and the lines extending fromthe end of the bars represent the standard deviation. The number ofcases in each group is as follows: 5 cases in the CNP gel preparation(30 μg/g) group, 5 cases in the CNP aqueous-solution preparation (100μg/ml) group, 7 cases in the CNP gel-base preparation (30 μg/g) group, 3cases in the CNP gel-base preparation (50 μg/g) group, 1 case in the CNPointment preparation (30 μg/g) group, 9 cases in the CNP ointmentpreparation (50 μg/g) group, 5 cases in the CNP ointment preparation(100 μg/g) group, 2 cases in the BNP gel-base preparation (30 μg/g)group, 5 cases in the BNP gel-base preparation (50 μg/g) group, 3 casesin the BNP aqueous-solution preparation (50 μg/ml) group, and 5 cases inthe ANP gel-base preparation (50 μg/g) group.

FIG. 20 is a graph showing the changes in the itching sensationsubjectively evaluated by the participants using visual analogue scalemethod, before and after application of the skin external-preparationcompositions comprising CNP or BNP of the present invention. The whitebars represent the levels before application, while the black barsrepresent the levels after application. The length of the barsrepresents the average, and the lines extending from the end of the barsrepresent the standard deviation. The number of cases in each group isas follows: 5 cases in the CNP gel preparation (30 μg/g) group, 5 casesin the CNP aqueous-solution preparation (100 μg/ml) group, 7 cases inthe CNP gel-base preparation (30 μg/g) group, 3 cases in the CNPgel-base preparation (50 μg/g) group, 1 case in the CNP ointmentpreparation (30 μg/g) group, 9 cases in the CNP ointment preparation (50μg/g) group, 5 cases in the CNP ointment preparation (100 μg/g) group, 2cases in the BNP gel-base preparation (30 μg/g) group, 5 cases in theBNP gel-base preparation (50 μg/g) group, 3 cases in the BNPaqueous-solution preparation (50 μg/ml) group, and 5 cases in the ANPgel-base preparation (50 μg/g) group.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The active ingredient of the skin external-preparation compositions ofthe present invention is C-type natriuretic peptide (CNP) or B-typenatriuretic peptide (BNP).

Here, the CNP means: CNP-22 composed of 22 amino acids, and CNP-53composed of 53 amino acids in which 31 amino acid residues are attachedto the N-terminal of the CNP-22, or derivatives thereof without anyparticular limitations provided that they possess CNP activity. TheseCNP-22, CNP-53, and their derivatives are all heretofore known, and canbe manufactured by chemical synthesis or genetic manipulations.

There are no particular limitations to the origin of CNP-22 and CNP-53,provided that they possess CNP activity, but the CNP derived frommammals including humans or birds is preferred, and more preferably, theCNP derived from humans, monkeys, mice, rats or pigs, and particularlypreferably, the CNP derived from humans.

The CNP derivatives means those having, in the amino acid sequences ofthe CNP-22 or CNP-53, deletion(s), substitution(s) or addition(s) of 1-5amino acids, more preferably 1-3 amino acids, and furthermore preferably1 or amino acids, while possessing CNP activity, or alternatively, thosehaving a sequence with a homology of 85% or more, preferably 90% ormore, and more preferably 95% or more with the amino acid sequence ofthe CNP-22 or CNP-53, while possessing CNP activity.

Replaceable amino acids are ideally substituted by conservative aminoacid substitution with amino acids having similar polarities andcharges. For example, nonpolar uncharged amino acids include glycine,alanine, valine, leucine, isoleucine, proline, etc.; aromatic aminoacids include phenylalanine, tyrosine, tryptophan; polar uncharged aminoacids include serine, threonine, cysteine, methionine, asparagine,glutamine, etc.; negatively-charged amino acids include asparaginicacid, glutamic acid; positively-charged amino acids include lysine,arginine, histidine. Thus, preferably amino-acid substitution is carriedout between conservative amino acids belonging to the same group. Here,when proline is to be replaced by another nonpolar uncharged amino acid,or when proline is to replace another nonpolar uncharged amino acid,then it should be noted that proline is not flexible in its spatialorientation. Similarly, when cysteine is to be replaced by another polaruncharged amino acid, or when cysteine is to replace another polaruncharged amino acid, then it should be noted that cysteine may form adisulfide bond with another cysteine.

CNP derivatives may include those amidated or methoxylated at aC-terminal, CNP modified with addition of polyethylene glycol or fattyacids, and, glycosylated or alkylated CNP, given that they have CNPactivity.

Thus, any heretofore known CNPs with CNP activity can be used in thepresent invention. Examples may include CNP derivatives disclosed in JPA 6-9688, CNP derivatives disclosed in U.S. Pat. No. 5,583,108, andCD-NP disclosed in U.S. Pat. No. 6,818,619. It is possible to easilytest the presence/absence of CNP activities using heretofore knownprocedures, such as by testing a growth inhibitory action on thevascular smooth muscle cells, or by examining the activity of cGMPproduction in the cells expressing NPR-B receptors.

While any of CNP-22, CNP-53 and their derivatives can be used as theactive ingredient of the present invention, CNP-22 with a smallermolecular weight is more preferable in terms of absorbability. CNP-22can be manufactured by chemical synthesis or genetic manipulations usinghuman CNP genes, and is also available from, for example, PeptideInstitute Inc. as CNP-22 (human).

CNP that can be used in the present invention includes: purifiednaturally occurring CNP, genetically engineered CNP manufactured usingknown genetic engineering procedures, CNP manufactured using knownchemical synthetic procedures (such as solid-phase peptide synthesis bya peptide synthesis machine). Basic methods including geneticengineering techniques, site-specific mutagenesis, and PCR, are commonlyknown or heretofore known, and are described in, for example, CurrentProtocols In Molecular Biology; John Wiley & Sons (1998), and JP A5-207891.

Here, the BNP refers to: BNP-26 composed of 26 amino acids, BNP-32composed of 32 amino acids, BNP-45 composed of 45 amino acids, or theirderivatives without any particular limitations provided that theypossess BNP activity. BNP can also be high molecular weight γ-BNP(molecular weight of approximately 13,000) which is formed by theremoval of the signal peptide from a BNP precursor. BNP-32 and theirderivatives are preferred. BNP-26, BNP-32, BNP-45, and their derivativesare heretofore known, and can be manufactured by chemical synthesis orgenetic manipulations.

There are no particular limitations to the origin of the BNP-26, BNP-32and BNP-45, provided that they possess BNP activity, but the CNP derivedfrom mammals including humans or birds is preferred, and the CNP derivedfrom humans, monkeys, mice, rats or pigs is more preferred, and the CNPderived from humans is particularly preferred.

The BNP derivatives means, those having, in the amino acid sequences ofBNP-26, BNP-32 or BNP-45, deletion(s), substitution(s) or addition(s) of1-5 amino acids, more preferably 1-3 amino acids, and furthermorepreferably 1 or amino acids, while possessing BNP activity, oralternatively, those having a sequence with a homology of 85% or more,preferably 90% or more, and more preferably 95% or more with the aminoacid sequence of BNP-26, BNP-32 or BNP-45, while possessing BNPactivity.

Replaceable amino acids are ideally substituted by conservative aminoacid substitution with amino acids having similar polarities andcharges. For example, nonpolar uncharged amino acids include glycine,alanine, valine, leucine, isoleucine, proline, etc.; aromatic aminoacids include phenylalanine, tyrosine, tryptophan; polar uncharged aminoacids include serine, threonine, cysteine, methionine, asparagine,glutamine, etc.; negatively-charged amino acids include asparaginicacid, glutamic acid; positively-charged amino acids include lysine,arginine, histidine. Thus, preferably amino-acid substitution is carriedout between conservative amino acids belonging to the same group. Here,when proline is to be replaced by another nonpolar uncharged amino acid,or when proline is to replace another nonpolar uncharged amino acid,then it should be noted that proline is not flexible in its spatialorientation. Similarly, when cysteine is to be replaced by another polaruncharged amino acid, or when cysteine is to replace another polaruncharged amino acid, then it should be noted that cysteine may form adisulfide bond with another cysteine.

BNP derivatives may include those amidated or methoxylated at aC-terminal of BNP, BNP modified with addition of polyethylene glycol orfatty acids, and, glycosylated or alkylated BNP, provided that they haveBNP activity.

Thus, any heretofore known BNP with BNP activity can be used in thepresent invention. Examples may include BNP derivatives disclosed in JPA 2007-525213, BNP derivatives disclosed in U.S. Pat. No. 6,028,055, BNPderivatives disclosed in U.S. Pat. No. 5,114,923, and BD-NP disclosed inU.S. Pat. No. 6,818,619.

It is possible to easily test the presence/absence of BNP activity usingheretofore known procedures, such as an examination of the activity ofcGMP production in the cells expressing NPR-A receptors.

While any of BNP-26, BNP-32, BNP-45 and their derivatives can be used asthe active ingredient of the present invention, BNP-32 is preferable interms of drug efficacy and availability.

BNP of the present invention can be manufactured by chemical synthesisor genetic manipulations using human BNP genes (for example, refer to JPA 5-207891, JP A 2007-525957, JP A 2007-525213), and BNP is alsocommercially available since it has already been launched.Alternatively, it is available from, for example, Peptide Institute Inc.as BNP-32 (human).

BNP that can be used in the present invention includes: purifiednaturally occurring BNP, genetically engineered BNP manufactured usingknown genetic engineering procedures, BNP manufactured using knownchemical synthesis procedures (such as solid-phase peptide synthesis bya peptide synthesis machine). Basic methods including geneticengineering techniques, site-specific mutagenesis, and PCR, are commonlyknown or heretofore known, and are described in, for example, CurrentProtocols in Molecular Biology; John Wiley & Sons (1998), and JP A5-207891.

When the term “CNP or BNP” is used herein, it refers to either CNP orBNP, as well as the chimeric peptides of CNP and BNP. That is, as usedherein, the term “CNP or BNP” refers to “CNP or BNP” which may be: achimeric peptide of CNP and BNP, in which CNP is CNP-22, CNP-53, or apeptide comprising any amino acid sequence with 5 or more consecutiveamino acids in the amino acid sequence having deletion, substitution, oraddition of any amino acid in the amino acid sequence of CNP-22 orCNP-53, and in which BNP is BNP-26, BNP-32, BNP-45, or a peptidecomprising any amino acid sequence with 5 or more consecutive aminoacids in the amino acid sequence having deletion, substitution, oraddition of any amino acid in the amino acid sequence of BNP-26, BNP-32or BNP-45, and wherein the chimeric peptide is the one that for a ringstructure by an intramolecular disulfide bond, and wherein the chimericpeptide is the one that has CNP activity or BNP activity; or aderivative of the chimeric peptide.

Here, there are no particular limitations to the origin of CNP-22 andCNP-53, provided that they possess CNP activity, but the CNP derivedfrom mammals including humans or birds are preferred, and morepreferably, CNP derived from humans, monkeys, mice, rats or pigs, andmost preferably, CNP derived from humans. Similarly, there are noparticular limitations to the origin of BNP-26, BNP-32 and BNP-45,provided that they possess BNP activity, but the BNP derived frommammals including humans or birds is preferred, and the BNP derivedhumans, monkeys, mice, rats or pigs is more preferred, and the BNPderived from humans is particularly preferred.

The derivatives of chimeric peptide of CNP and BNP mean those whichhave, in the amino acid sequences of the chimeric peptide of CNP andBNP, a deletion, addition or substitution of preferably 1-5 amino acids,more preferably 1-3 amino acids, and furthermore preferably 1 or 2 aminoacids, while possessing CNP or BNP activity.

Replaceable amino acids are ideally substituted by conservative aminoacid substitution with amino acids having similar polarities andcharges. For example, nonpolar uncharged amino acids include glycine,alanine, valine, leucine, isoleucine, proline, etc.; aromatic aminoacids include phenylalanine, tyrosine, tryptophan; polar uncharged aminoacids include serine, threonine, cysteine, methionine, asparagine,glutamine, etc.; negatively-charged amino acids include asparaginicacid, glutamic acid; positively-charged amino acids include lysine,arginine, histidine. Thus, preferably amino-acid substitution is carriedout between conservative amino acids belonging to the same group. Here,when proline is to be replaced by another nonpolar uncharged amino acid,or when proline is to replace another nonpolar uncharged amino acid,then it should be noted that proline is not flexible in its spatialorientation. Similarly, when cysteine is to be replaced by another polaruncharged amino acid, or when cysteine is to replace another polaruncharged amino acid, then it should be noted that cysteine may form adisulfide bond with another cysteine.

The derivatives of chimeric peptide of CNP and BNP may include thoseamidated or methoxylated at C terminal of the of chimeric peptide of CNPand BNP, those modified with addition of polyethylene glycol or fattyacids in the chimeric peptide of CNP and BNP, and, glycosylated oralkylated chimeric peptide of CNP and BNP, provided that they have CNPor BNP activity.

Thus, it is possible to use any heretofore known chimeric peptides ofCNP and BNP or derivatives thereof in the present invention, on thecondition that they possess CNP or BNP activity.

Presence/absence of CNP or BNP activity can be easily tested usingheretofore known procedures, such as an examination of the activity ofcGMP production in the cells expressing NPR-A receptors or in the cellsexpressing NPR-B.

The chimeric peptides of CNP and BNP and their derivatives of thepresent invention can also be manufactured by chemical synthesis or bygenetic manipulations.

Dermatitis is an inflammation of the skin, characterized by commonsymptoms such as erythema, infiltrative erythema, lichenified lesions,scales, adhesion of crusts, eczema, abrasion, excoriations, prurigonodularis, papules, erosions, infiltration, vesicles, edema, etc., andin particular, symptoms such as itching, blisters, reddening, swelling,feeling of oozing, scabs, scale formation, etc.

The skin external-preparation composition of the present invention canbe applied to dermatitis patients with inflammation, preferablysuffering from, but not limited to; atopic dermatitis, dermatitis thatled up to steroid dermatitis, steroid-resistant dermatitis, dermatitiswhich cannot be treated with tacrolimus, chronic dermatitis,erythroderma, eczema, contact dermatitis, seborrheic dermatitis,autosensitization dermatitis, stasis dermatitis, urticaria, drugeruption, cutaneous angiitis, prurigo, pruritus cutaneus, erythema,psoriasis, rosacea, rosacea-like dermatitis, lichen planus, follicularkeratosis, and more preferably from atopic dermatitis, dermatitis thatled up to steroid dermatitis, steroid-resistant dermatitis, dermatitiswhich cannot be treated with tacrolimus, chronic dermatitis, eczema,erythroderma, rosacea, rosacea-like dermatitis, and psoriasis, and evenmore preferably from atopic dermatitis, steroid-resistant dermatitis,chronic dermatitis, eczema, erythroderma, rosacea, rosacea-likedermatitis, and psoriasis, and most preferably from atopic dermatitis.

A skin external-preparation composition means a composition externallyand directly applicable to the skin, and more specifically refers to atherapeutic agent for dermatitis or skin texture-improving agent. Thereare no particular limitations in its dosage form, but preferably it isan ointment, gel, cream, lotion, solution, spray, or patch, and morepreferably, an ointment, gel, cream or solution. These dosage forms areparticularly suited for therapeutic agents for dermatitis. When the skinexternal-preparation composition of the present invention is to be usedas a skin texture-improving agent, the preferred dosage forms are thefollowing; cream, foam, skin lotion, facial mask, skin-softening water,skin emulsion, foundation, makeup base, essence, soap, liquid cleanser,bath agent, sun-block cream, suntan oil or spray-type liquidpreparation. When the skin external-preparation composition of thepresent invention is used as a skin texture-improving agent, it may be askin-care cosmetic or a quasi drug.

In terms of indication, the skin external-preparation composition of thepresent invention as a therapeutic agent for dermatitis can be appliedto dermatitis associated with at least one of the rash symptoms selectedfrom: erythema, infiltrative erythema, lichenified lesions, scales,adhesion of crusts, eczema, abrasion, excoriations, prurigo nodularis,papules, erosions, infiltration, vesicles, and edema. These symptoms arethe symptoms observed in patients with atopic dermatitis.

Atopic dermatitis mentioned above is defined by the JapaneseDermatological Association as a dermatitis for “which exacerbations andremissions recur, being mainly characterized by pruritic eczema, inwhich most of the patients have atopic diathesis”.

That is to say, atopic dermatitis is a chronic eczema associated withitching that occurs in those who have an allergic predisposition. It isa typical inflammatory skin disease showing the conditions wherein thesymptoms accompany itching, the rashes are eczematous lesions, whichreddens as an acute lesion (erythema), oozing eruptions (papules andserous papules) are formed, then the skin peeled off and crusts areformed (scales and crusts). The chronic pathological lesions includethickening and hardening of the skin (lichenification), and formation ofhard lumps (prurigo). Rashes have a tendency to develop on the forehead,around the eyes, around the mouth, on the neck, around the joints suchas the elbows, knees and wrists, on the dorsal and abdominal regions,and they are often characterized by a symmetrical distribution. Ininfantile atopic dermatitis, the lesions generally appear on the headand face first, and occasionally spread to the trunk and four limbs,whereas in puberty and adulthood, severe rashes tend to occur in theupper body (face, neck, chest and back). Atopic dermatitis also has atendency to be chronic or recurrent lasting over 6 months, or 2 monthsin infants.

The skin external-preparation of the present invention, in particularthe therapeutic agent for dermatitis, is both safe and effective as atherapeutic agent for such atopic dermatitis which has conventionallybeen regarded as a difficult-to-treat disease.

Dermatitis that led up to steroid dermatitis refers to dermatitis,wherein it develops a group of adverse side effects due to the long-termcontinuous use of steroid external drugs. In particular, dermatitis inwhich a rebound phenomenon occurs upon withdrawal from steroid externaldrugs is called steroid dermatitis, and steroid rosacea is a typicalexample of steroid dermatitis.

Steroid-resistant dermatitis refers to dermatitis with decreasedresponsiveness to the steroid external preparations resulting fromlong-term use of the steroid external preparations. Steroid-resistantdermatitis tends to occur when steroid external preparations are usedfor a long time for the treatment of atopic dermatitis.

Dermatitis to which tacrolimus is not applicable, means dermatitissuffered by pregnant women, infants below the age of 2, patients withoozing ulcerated regions or scratches, and women who are breast-feeding.In cases of dermatitis in immune-compromised patients and patients witha kidney disorder, caution needs to be taken with the use of tacrolimus.Hence, these dermatitides can also be regarded as the dermatitis towhich tacrolimus is not applicable.

Chronic dermatitis is a dermatitis that has become chronic andintractable, and includes atopic dermatitis and contact dermatitiscaused by an allergy or an irritation by the substance that contacts theskin.

Psoriasis is a chronic and repeatedly recurrent skin disease,characterized by one or several erythematous rashes (erythema) andsilvery-white squamate scales.

Erythroderma, also called exfoliative dermatitis, commonly develops fromatopic dermatitis or eczema in elderly patients, and it is a dermatitischaracterized by skin flush over the whole body accompanied bydesquamation. Diffuse erythema is observed over the whole body or onwide areas of the skin. Erythroderma includes erythrodermaposteczematosa and erythroderma secondary to dermatoses, toxicerythroderma, infantile desquamative erythroderma, and paraneoplasticerythroderma. Of these, erythroderma posteczematosa refers to a statecalled erythroderma that shows skin flush and desquamation over thewhole body, caused by generalization of eczema due to long-term externalapplication of inappropriate steroids or due to an increase of severity.Erythroderma is a significantly intractable disease with systemicsymptoms, which is usually associated with itching, and as systemicsymptoms, associated with failure of thermoregulation such as fever,chills, and shivering, hypoproteinemia and edema occurring withdesquamation, electrolyte abnormality due to loss of water from theexfoliated skin, swelling of lymph nodes, a general feeling of malaise,and loss of body weight. Internal or external application of steroids isadopted for the treatment; however, because of its significantlyintractable nature, symptoms may immediately relapse or even worsen asrebound phenomena upon discontinuation of medication.

Rosacea is a persistent skin disease, which typically producesconditions in which reddening and small pustules occur on the centralarea of the face, and in which blood vessels are fairly visible.

Rosacea-like dermatitis is characterized by many red papules, diffusedskin flush and desquamation, and is often seen in adult females. Itsmajor development factor is a long-term external application ofsteroids, and it is one of the representative side effects of steroidexternal preparations; for its treatment, rebound of symptoms is severe,and therapeutic technique and the patients′ patience are required forgetting over it.

Prurigo often induces rashes as symptoms in addition to itching. Majorcauses of prurigo include parasites such as Sarcoptes scabiei, mites andlice, insect bites, urticaria, atopic dermatitis, allergic dermatitisand contact dermatitis, etc.

Erythema multiforme is a repeatedly recurrent dermatosis characterizedby red raised skin lesions, presenting rashes that look like shootingtargets. Symptoms of erythema multiforme often appear as a result ofinfection with herpes simplex viruses. In many cases, Erythemamultiforme develops suddenly, and presents with red rashes (erythema) onthe arms, legs and face as symptoms. Erythema presents with symptoms ina concentric fashion like a shooting target, sometimes associated withblisters.

Erythema nodosum is an inflammatory disease with tender red lumps(nodules) under the skin. Quite frequently it appears as a symptom ofother diseases or as hypersensitivity to drugs. Young adults, inparticular females are prone to erythema nodosum. It relapses repeatedlyfor several months to several years. Erythema nodosum may occur frominfection with bacteria, fungi, or viruses.

Lichen planus is a skin disease with recurrent itching. Its symptomsinclude occurrence of small red or purple raised rashes. Initially therashes are individually separated, then in many cases, plural rashes arefused to form papules associated with rough, scaly and dry skin.

Lichen pilaris is also called as follicular keratosis. It is a diseasein which the orifice of the hair follicle is clogged with dead cells(cuticle) that have been sloughed off from the upper layer of the skin.

Contact dermatitis includes irritant contact dermatitis and allergiccontact dermatitis. The former is an inflammation of the skin caused bydirect contact of the skin with a specific irritant, such as acid,alkali and solvent, etc. The rash is associated with strong itching, andaffected regions are limited, often having a clear boundary with normalskin.

The therapeutic agent compositions for dermatitis of the presentinvention can be formulated using widely used technologies. Examples oftheir formulation include external preparation, injectable preparation,oral preparation, and nasal preparation. In the case of oralpreparation, enteric-coated preparations are preferred in order to avoidpeptide degradation in the stomach. Examples of enteric-coatedpreparations include preparations wherein an enteric-substance is coatedon capsules, tablets, or granules. In general, since peptide drugs arerapidly metabolized and easily excreted from the body, they can bemodified with polyethylene glycol (PEGylation) in order to prolong theirhalf life without affecting the biological activities, and to decreaseantigenicity.

The preferred dosage form of the therapeutic agent compositions fordermatitis of the present invention is external preparations(transdermal absorption preparations) such as gel preparations, ointmentpreparations, liquid preparations, etc.

External preparations are not particularly limited as long as thepresent agent can be directly applied, sprayed, or attached to thedesired region of the skin (affected area). The dosage form of thetherapeutic agent composition for dermatitis of the present invention ispreferably an external preparation such as an ointment preparation, gelpreparation, cream preparation, lotion preparation, liquid preparation,spray preparation, or patch preparation, and particularly preferably,from the viewpoint of ease of application, an ointment preparation, gelpreparation, cream preparation or liquid preparation, and morepreferably a gel preparation, ointment preparation or liquid preparationconsisting of an aqueous solution.

These external preparations can be easily obtained in accordance withheretofore known or well-known methods, by blending a pharmaceuticallyacceptable base and, as necessary, various additives, with CNP or BNP asan active ingredient or a principal agent.

A gel preparation (suspension base) may be a hydrous gel, an anhydrousgel, or a gel with a low water content comprising a gel-forming materialthat can swell. It may also be a hydrogel base or a lyogel base, andpreferably a transparent hydrogel having an inorganic or organic polymeras a base. Similar to those preparations comprising an oil or fatcontent, the gel itself is not absorbed by the skin. Hydrogel bases haveno fat, have a consistency similar to that of ointment, and aim atincreasing the transdermal absorbability of drugs. Lyogel bases aregelled by suspending stearyl alcohol, etc. in propylene glycol, and theyhave excellent transdermal absorbability and hygroscopicity.

The gel preparation (suspension base) of the present invention may be agel preparation (suspension base) manufactured by homogenouslydispersing CNP or BNP as an active ingredient into a hydrophilic gelbase comprising carboxy vinyl polymer, sodium polyacrylate, sodiumpolyacrylate, (vinyl methyl ether/ethyl maleate) copolymer,polymethacrylate, propylene glycol, etc. Examples of such gelpreparations (suspension bases) include gel preparations (suspensionbases) wherein ingredients are homogeneously dispersed in acommercially-available long-lasting water-retention agent, such asLubrajel NP, Lubrajel CG, Lubrajel DV, Lubrajel MS, Lubrajel OIL,Lubrajel TW, Lubrajel DS, which are commercially-available productsavailable from ISP Japan, Ltd., etc.

As used herein, “gel preparation” refers to gel preparations (suspensionbases) prepared in accordance with Example 2, comprising dipotassiumglycyrrhizinate, allantoin, pyridoxine hydrochloride, xanthan gum, andvitamin E. As used herein, “gel-base preparation” refers to gelpreparations (suspension bases) prepared in accordance with Examples 7,13 or 14, which differ from the “gel preparation” in that the gel-basepreparation does not comprise dipotassium glycyrrhizinate, allantoin,pyridoxine hydrochloride, xanthan gum, and vitamin E. The “gelpreparations” (suspension base) of the present invention include both“gel preparations” and “gel-base preparations.”

A liquid preparation means those wherein an active ingredient consistingof CNP or BNP is dissolved in a base such as alcohol, propylene glycol,polyethylene glycol or water. Preferably, it means a liquid preparationconsisting of an aqueous solution wherein either CNP or BNP is dissolvedin saline. In the aqueous solutions, a small amount of an organic basesuch as alcohol, propylene glycol, polyethylene glycol, etc. may bemixed, in addition to saline.

At this time, in order to ensure the extent of bioavailability and toprovide more effective liquid preparations, in other words, with the aimof improving the extent of bioavailability upon subcutaneous injectionof a bioactive peptide comprising CNP or BNP, it is possible to make thepH of the solution to be 3.0-7.0 by making an acid solution wherein oneor more from the group consisting of butyric acid, lactic acid,phosphoric acid, glycine, citric acid, hydrochloric acid, propionicacid, butyric acid, benzoic acid, and salts thereof are combined withthe bioactive peptide CNP or BNP as the active ingredient, or by makinga polar organic solution wherein one or more from the group consistingof alcohols, and/or N-methyl-2-pyrrolidine, dimethylformamide, dimethylsulfoxide, and methylparaben are combined with the bioactive peptide CNPor BNP as the active ingredient.

An ointment preparation may be either a grease base or a water-solublebase, and both can be easily obtained in accordance with heretoforeknown methods. A grease base such as vaseline has little irritation andis odorless, which is superior in protective action of the skin,softening action, crust-removal action, formation of granulation tissue,and epithelialization-stimulating action. A water-soluble base is anointment having a macrogol base as the main ingredient, and has a strongaction to absorb and remove aqueous secretions.

A cream preparation (emulsion base) may be an oil-in-water base (O/W)(vanishing cream) or a water-in-oil base (cold cream). An oil-in-waterbase has a smaller amount of oil-soluble component than water-solublecomponent. It has an advantage that the white color of a cream appearsto disappear upon application. It extends well and feels good uponapplication to sweaty skin, thus it is cosmetically superior. Inaddition, it also has a good absorbability into the skin, thus isapplicable to chronic hypertrophic lesions. A water-in-oil base has asmaller amount of water-soluble component than oil-soluble component,and is also called a cold cream because it has a cooling action uponapplication by extending over the skin.

A lotion preparation means a liquid external preparation wherein CNP orBNP is dissolved or homogeneously dispersed in a liquid. Since ointmentsand creams tend to adhere to the hair, lotions are suitable for use onthe head hair region, etc. The form of lotions may be any of asuspension lotion base, an emulsion lotion, and a solution-type lotionbase.

In a patch preparation, a component comprising CNP or BNP is adsorbed toa patch, thereby stimulating absorption of the drug by utilizing theairtight characteristic of the patch. Upon application of the patch,scratching can be prevented.

A spray preparation refers to those wherein CNP or BNP is made into asolution, which is then sprayed by gas pressure. Sprays are convenientwhen applied to wide areas.

Thus, the therapeutic agent composition for dermatitis of the presentinvention is a transdermal external preparation comprising anappropriate amount of CNP or BNP and various bases, as well as additivesas necessary. To exert drug effects as an external preparation, it isimportant that the concentration of the active ingredient (CNP or BNP)applied to the skin surface can reach an effective concentration ataffected lesions, and that the concentration can be maintained.Accordingly, dosage forms and bases can be appropriately selecteddepending on the symptoms and patient.

Additives may be appropriately used depending on objectives. Asadditives, the following may be used.

Vaseline: Vaseline can be used as a base for ointment preparations.Viscosity and consistency vary with temperature, and its hardnessdiffers between winter and summer. Vaseline is one of the safest bases.There are yellow vaseline, and white vaseline with a higher purity; bothcan be used.

Propylene glycol: Propylene glycol can be used as a solvent,solubilizing agent, or base for drugs.

Paraffin: Paraffin can be used when adjusting the viscosity/consistencyof ointment preparations. Since its emulsification is relatively-easy,paraffin may also be used as an oil base for production of creams.

Bees wax (white beeswax): Bees wax is a processed wax of the honeycomb,which can be used as “Japanese Pharmacopoeia” simple ointment byblending with plant-derived fat and oil. White beeswax is a bleachedproduct of bees wax to improve color and odor.

Macrogol: Macrogol is a mixture of polyethylene glycols with differentmolecular weights. It has good drug solubility and mixingcharacteristic, absorbs water well; thus is suitable for adsorption andelimination of eluate from mucosa and affected area.

Stearyl alcohol: Stearyl alcohol can be used for emulsion lotions.

Isopropanol: Isopropanol can be used as a solvent or solubilizing agent,etc.

Benzyl alcohol: Benzyl alcohol can be used as a solubilizing agent andpreservative, etc.

Parahydroxybenzoate esters (parabens): Parahydroxybenzoate esters can beused as an antiseptic agent, preservative, and stabilizer.

Gelled hydrocarbon: Gelled hydrocarbon is generally called “Plastibase”,which is made by making liquid paraffin into a gelled (semi-solid) stateusing polyethylene.

Citric acid, sodium citrate: Citric acid and sodium citrate can be usedas buffering agents or pH adjusters.

Squalene: Squalene is used as a base, and has slightly less oilyfeeling, being less sticky, than liquid paraffin. Similar to creams,squalene can also be used widely for emulsion lotions.

Lanolins: Lanolins are fats and oils obtained from sheep's wool;although lanolins have drawbacks in terms of color and odor, they areeffective for improving the softness of the skin.

Glycerin: Glycerin can be blended in creams, etc. as a moisturizingagent.

Polyoxyethylene hardened castor oil: This can be used as an emulsifyingagent, solubilizing agent, etc.

Sorbitan fatty acid ester, glycerin fatty acid ester: They can be usedas emulsifying agents, etc.

The therapeutic agent for dermatitis of the present invention mayfurther comprise moisturizing agents (skin softening agents) andsymptom-relieving agents, etc. as described below.

Moisturizing agent (skin softening agent): Moisturizing agents providemoisture and oil content to the skin. Moisturizing agents are mosteffectively used when the skin is already moisturized, for example, justafter taking a bath or shower. Components contained in moisturizingagents include glycerin, mineral oil, and vaseline, etc. The form andtype of moisturizing agents include lotion preparations, creampreparations, ointment preparations and bathing oils, etc. Thosecomprising urea, lactic acid and glycolic acid have superiormoisturizing effects.

Symptom-relieving agent: Skin diseases are often accompanied by itching.Itching and mild pain can be reduced by blending a sedative drug,specifically, chamomile, eucalyptus, camphor, menthol, zinc oxide, talc,glycerin, and calamine, etc. To suppress itching due to an allergy,antihistamine agents such as diphenhydramine may be comprised.

As such, when manufacturing the therapeutic agents for dermatitis of thepresent invention, the following various agents may be arbitrarilyblended in combination: base, moisturizing agent, ultraviolet absorbingagent, alcohols, chelates, pH adjuster, antiseptic agent, thickeningagent, coloring agent, flavor, filling agent, excipient, disintegratingagent, filler, binding agent, coating agent, solubilizing agent,suspending agent, buffer, stabilizing agent, preservative, surfactant,antioxidative agent, dispersing agent, emulsifying agent, dissolvingagent, solubilizer, etc. In addition to CNP or BNP, which is theprincipal agent, various drugs such as antiphlogistic analgetics,sterilizing agents, vitamins, skin softening agents, etc. may beappropriately blended as necessary.

When the skin external-preparation composition of the present inventionis used as a skin texture-improving agent, it can be used as a skin-carecosmetic or quasi drug; specific usage forms include cream, foam, skinlotion, facial mask, skin-softening water, skin emulsion, foundation,makeup base, essence, soap, liquid cleanser, bath agent, sun-blockcream, suntan oil, or spray-type liquid preparation. These may be easilyproduced by application of well-known or heretofore known formulationtechnologies.

Next, as representative examples of preparations of the therapeuticagent composition for dermatitis of the present invention, production ofaqueous-solution preparations as a liquid preparation and gelpreparation will be described.

In the present invention, one of the preferred external preparations isan aqueous-solution preparation.

Such an aqueous-solution preparation, can be prepared for example, as aliquid preparation with a CNP or BNP concentration of 1-1000 μg/g, bydissolving 0.01-10 mg of human CNP-22 (Peptide Institute, Inc.) or humanBNP-32 (Peptide Institute, Inc.) as the principal agent into 10 ml ofsaline. Here, since the specific gravity of water is 1, the CNP or BNPconcentration in this case is 1-1000 μg/g by weight ratio. When theblending ratio is 1 μg/g or less, effects are not sufficient, andsufficient effects can be obtained with blending at a ratio of no morethan 500 μg/g. Concentrations of CNP or BNP in an aqueous-solutionpreparation are preferably 1-500 μg/g, more preferably 10-500 μg/g,furthermore preferably 20-200 μg/g, and particularly preferably 30-100μg/g.

Gel preparations can be obtained by, in accordance with heretofore knownor well-known methods, dissolving an appropriate amount of CNP or BNPinto distilled water or saline to make an aqueous solution, and bymixing and stirring a heretofore known or well-known gelling agent withthe solution. The final concentration of the CNP or BNP in the gelpreparation should be prepared to be preferably 1-500 μg/g, morepreferably 10-500 μg/g, furthermore preferably 20-200 μg/g, andparticularly preferably 30-100 μg/g.

Examples of the gelling agent consisting of macromolecular inorganiccomponents include hydrous or water-absorbing silicates, such asaluminum silicate, bentonite, magnesium aluminum silicate, and colloidsilica. As the gelling agent consisting of macromolecular organicsubstances, natural, semi-synthetic, or synthetic polymers may be used.Examples of natural and semi-synthetic polymers include, polysaccharidessuch as cellulose etc., starch, tragacanth, gum arabic, xanthan gum,agar, gelatin, alginic acid and its salts, for example, sodium alginateand its derivatives, lower alkyl cellulose, for example, methylcellulose or ethyl cellulose, carboxy- orhydroxyl-lower-alkyl-cellulose, for example, carboxymethyl cellulose, orhydroxypropyl cellulose, etc. Examples of synthetic gelling agentsinclude polyvinyl alcohols, polyvinyl pyrrolidones, polyacrylic acids orpolymethacrylic acids, etc. Only one kind of these gelling agents, or amixture of two or more kinds of these may be used.

As necessary, a transdermal absorption aid may be added. Examples of thetransdermal absorption aid include, for example, acetic acid, sodiumacetate, limonene, menthol, salicylic acid, hyaluronic acid, oleic acid,N,N-diethyl-m-toluamide, n-butyl stearate, benzyl alcohol, isopropylmyristate, isopropyl palmitate, polypropylene glycol, crotamiton,diethyl sebacate, N-methyl pyrrolidone, N-ethyl pyrrolidone, laurylalcohol, etc. In addition, an antiseptic agent and an antioxidant may beadded as necessary.

The concentration of CNP or BNP in the therapeutic agent composition fordermatitis of the present invention may be appropriately selectedaccording to symptoms, age, and dosage form, etc. The preferableconcentration of CNP or BNP is, for external preparations such as liquidpreparations, gel preparations and lotion preparations, etc., 1-500μg/g, more preferably 10-500 μg/g, furthermore preferably 20-200 μg/g,and particularly preferably 30-100 μg/g. For younger patients orpatients with sensitive skin, the use of 20-100 μg/g concentration ispreferred. The concentration of CNP or BNP in a gel preparation andointment preparation is preferably 1-500 μg/g, more preferably 10-500μg/g, and furthermore preferably 20-200 μg/g, and particularlypreferably 30-100 μg/g. The concentration of CNP or BNP in a liquidpreparation is preferably 1-500 μg/ml, more preferably 10-500 μg/ml,furthermore preferably 20-200 μg/ml, and particularly preferably 30-100μg/ml. Here, since the specific gravity of the solution used in theliquid preparation of the present invention is almost 1, when theconcentration of CNP or BNP in the liquid preparation is indicated inthe units of μg/g, it is equivalent to the indication in the units ofμg/ml for the concentration of CNP or BNP.

The administration of the therapeutic agent composition for dermatitisof the present invention differs depending on symptoms, age and dosageform, etc.; however, administration is normally once or twice a day, andthe duration of administration is from 1 to 10 days.

EXAMPLE 1

Diagnosis and Evaluation of Subjects

First, prior to administration of the CNP preparation or BNP preparationof the present invention, subjects were diagnosed and evaluated. Themethods for diagnosis and evaluation of the subjects are as follows.

1. Diagnosis of Subjects:

All the subjects were patients for whom administration of existingexternal drugs such as steroids, etc. was ineffective. The diagnosis andtreatment of these subjects were performed by the present inventor as amedical doctor.

2. Evaluation of Symptoms:

The evaluation of symptoms of atopic dermatitis was performed inaccordance with, in principle, “2005 Guideline for the Treatment ofAtopic Dermatitis by the Scientific Research Division of the Health andWelfare Ministry of Japan” (hereinafter, simply referred to as“Guideline 2005”) by the classification into four levels as shown inTable 1.

TABLE 1 Evaluation of symptoms Mild Only mild rash is observedregardless of size. Moderate Rash with severe inflammation is observedin less than 10% of the body surface area. Severe Rash with severeinflammation is observed over 10% or more and less than 30% of the bodysurface area. Most Rash with severe inflammation is observed over severe30% or more of the body surface area. “Mild rash”: means lesions mainlycharacterized by mild erythema, dryness, and desquamation. “Rash withsevere inflammation”: means lesions associated with erythema, papules,erosions, infiltration and lichenification, etc.

In addition, the severity level of the rash at each region wasdetermined in accordance with “Guidelines for Management of AtopicDermatitis by the Japanese Dermatological Association” (hereinafter,simply referred to as the “Dermatological Association Guideline”), asshown in Table 2.

TABLE 2 Severity level of rash Minor Mainly characterized by drysymptoms with a little inflammation symptoms. Mild Mainly characterizedby dryness and mild erythema and scales, etc. Moderate Mainlycharacterized by up to mild erythema, scales, a small number of papulesand abrasion, etc. Severe Mainly characterized by erythema with severeswelling/edema/infiltration/or lichenification, many papules, severescales, adhesion of crusts, vesicles, erosions, numerous excoriations,and prurigo nodularis, etc.

The severity level of the rash of each region was also determined usingSCORAD (SCORing of Atopic Dermatitis) index, which was proposed by theEuropean Task Force on Atopic Dermatitis and is widely used throughoutthe world. With the SCORAD index, a severity level is determined by thesummation of rating scores for each of (A) extent %, (B) intensity ofrash, (C) subjective symptoms. In the evaluation of the present study,the intensity of rash (B) was evaluation with the following 4 levels: 0:none, 1: mild, 2: moderate, 3: severe, with respect to 6 items:erythema, edema/papulation, oozing/crusting, excoriation,lichenification, and dryness. Since the regions of application werelimited to parts of the body instead of the whole body, the total scorewas not determined in accordance with a specified calculation formula ofthe severity-level classification by SCORAD. Instead, a score, which isa simple summation of the rating scores of the 6 items of intensity ofrash at the region of application before and after the application, wasused. Since choice of the external therapeutic method, which is aprimary therapy, is determined based on the severity of each rash, theseverity level of each rash is the most important factor in selectingexternal therapy as well as in predicting therapeutic outcomes. Detailsof the SCORAD index are described, for example, in C. Gelmetti and C.Colonna, Allergy, Vol. 59, Supplement 78, p. 61, 2004.

3. Test Method:

In general, in order to evaluate the effects of external preparations onindividual cases, a right/left comparative method is suitable. It is amethod wherein, the therapeutic effects of active ingredients areidentified by, for example, applying an external preparation comprisingthe active ingredient to be tested is applied to the left side of theaffected region, and an external preparation without the activeingredient is applied to the right side. The preparations of the presentinvention were tested by right/left comparative method in preliminarytests. However, considering the medical ethics, preliminary tests by theright/left comparative method were limited to a minimum degree, and whenpreliminary tests were not performed, therapeutic effects were evaluatedby the comparison between before and after application.

EXAMPLE 2

1. Production of CNP Gel Preparation

0.1 g of methyl parahydroxybenzoate (product name: Mekkins M, Ueno FineChemicals Industry), 0.2 g of phenoxyethanol, 3.0 g of 1,2-pentanediol,6.0 g of concentrated glycerin, 0.1 g of dipotassium glycyrrhizinate,0.1 g of allantoin, and 0.05 g of pyridoxine hydrochloride are added anddissolved in 75.72 g of purified water. Then 6.0 g of Lubrajel fromShowa Denko K.K. (a mixture consisting of 4.674 g of purified water,0.12 g of carboxy vinyl polymer, 0.006 g of sodium polyacrylate, and 1.2g of glycerin), 0.44 g of carboxy vinyl polymer (product name: Carbopol940, Lubrisol Advanced Materials, Inc.), and 8.00 g of 1% xanthan gumsolution (product name: Keltrol T, CP Kelco) were added to this solutionand stirred and mixed, then 0.04 g of natural vitamin E was furtheradded to make a homogenous mixed solution. Finally, 0.25 g of potassiumhydroxide was added for neutralization, and the solution wassufficiently stirred to form a gelled state to obtain a gel preparation.

The CNP gel preparation was prepared as follows: 3 mg of human CNP-22(Peptide Institute, Inc.) as the principal agent was dissolved in 3 mlof saline, and 100 μl of the resulting solution was diluted with 400 μlof saline to adjust the concentration to be 200 μg/ml, then 1.5 ml ofthe resulting solution was mixed and stirred in 8.5 g of the above gelpreparation. The CNP concentration of thus-obtained gel preparation is30 μg/g.

EXAMPLE 3

2. Production of CNP Aqueous-Solution Preparation

The CNP aqueous-solution preparation was prepared as follows: 3 mg ofhuman CNP-22 (Peptide Institute, Inc.) as the principal agent wasdissolved in 3 ml of saline, and 100 μl of the resulting solution wasdiluted with 900 μl of saline to prepare the aqueous-solutionpreparation with a CNP concentration of 100 μg/ml. CNP aqueous-solutionpreparations with a CNP concentration of 50 μg/ml and 200 μg/ml wereprepared similarly.

EXAMPLE 4

3. Diagnosis of Subjects:

Prior to administration of the CNP gel preparation and CNPaqueous-solution preparation of the present invention, history takingfrom the subjects, scratch tests for allergens, and a diagnosis wereconducted. Table 3 (subjects 1-5) and Table 5 (subjects 6-10) show theresults of the subjects′ history taking and diagnosis, i.e., sex, age,onset and course of disease, family history, past history, scratch testresults, diagnostic findings, and symptom evaluation based on “Guideline2005” of the subject in each case.

EXAMPLE 5

4. Therapeutic Effects on Subjects

Therapeutic effects of the CNP gel preparation and CNP aqueous-solutionpreparation of the present invention are shown in Table 4 (subjects 1-5)and Table 6 (subjects 6-10). In Tables 4 and 6, “itching sensation”represents a comparison of the itching sensation evaluated using thevisual analogue scale method before and after treatment. Similarly,“non-recurrence period” refers to the period after discontinuation ofthe treatment by the preparation of the present invention subsequent toimprovement of symptoms, in which relapse of the symptoms did not occur.In order to evaluate objectively, photographs before and afterapplication of CNP preparations were taken for all cases. Of these,photographs of some cases are shown in the figures.

TABLE 3 Diagnosis prior to application of CNP gel preparation. SubjectSubject 1 Subject 2 Subject 3 Subject 4 Subject 5 Case Case 1 Case 6Case 5 Case 9 Case 10 Sex Female Female Male Female Male Age 21 yearsold 39 years old 24 years old 9 months old 32 years old Onset andDeveloped in Developed at 19 Have repeated Developed at 10 Developed incourse of infancy, having years of age, chronic days after birth;infancy, having disease recurrent having recurrent recurrentgeneralized-type recurrent eczema eczema with eczema with dermatitiswith recurrent eczema with itching, itching. itching. She is itchingfrom with itching. The generalized type; in the state of elementary facebecomes he is in a state erythroderma school. occasionally full oferythroderma posteczematosa Generalized of excoriations posteczematosa;presumably due to type; symptoms due to scratching chills, skin flushlong period of on the face tend the skin all the and scales are steroidexternal to worsen upon time. Horse oil observed. therapy, and skin useof organic has been used for flush and solvents and moisturization.desquamation are lack of sleep. observed over the whole body. FamilyMother and Mother; Atopic Mother; Father; Atopic None history elderbrother; dermatitis Bronchial asthma dermatitis Atopic dermatitis,allergic rhinitis Past Allergic Child asthma Allergic Child asthma,history rhinitis rhinitis Allergic rhinitis Scratch House dust: 5+ Housedust: 3+ House dust: 2+ House dust: 1+ House dust: 2+ test Mite: 5+Mite: 2+ Mite: 2+ Mite: 2+ Mite: 3+ Cedar: 1+ Egg white: 2+ Orchardgrass: Egg yolk: 2+ 2+ Ragweed: 1+ Diagnostic Infiltrative InfiltrativeMild-to-severe Erythema, Infiltrative findings erythema with erythemawith level erythema, papules, scales erythema with itching itchingscales and and numerous itching associated associated with associatedwith excoriation are excoriations are with sleep sleep sleep observedover observed over the disturbance, disturbance, disturbance, the wholebody. whole body. lichenified lichenified lichenified lesions, andlesions, and lesions, and erythema are erythema are erythema areobserved over the observed over observed over the whole body. In thewhole body. whole body. particular on the severe scales and upper limbs,infiltrative infiltrative erythema with erythema with strong burningstrong burning sensation are sensation is observed on the repeatedlyface. worsened. Symptoms of Rash on the Rash on the face Rash on theback Rash on the both Rash on the application face and neck is mainly ismainly forearms is forearms is mainly regions is mainly characterized bycharacterized by mainly characterized by characterized severe severeswelling, characterized by severe swelling, by severe infiltration,erythema with erythema, infiltration, infiltration, erythema, and edema,and papules, scales, erythema, scales, and erythema numerous scales. andnumerous papules, erosions with edema. excoriations. excoriations. andnumerous excoriations. Effects of Steroid She is in the Steroid external(Parents) desire He is in the state steroid external drug state oftherapy relieves use treatment of erythroderma external had no effectserythroderma the infiltrative without steroids. posteczematosa drug onerythema, posteczematosa erythema on the presumably due to infiltrativepresumably due to back and face long-term steroid erythema and steroidexternal only external therapy. scales on the therapy. insufficiently,face and neck. and the symptoms relapse soon. Evaluation Most severeMost severe Severe Most severe Most severe of symptoms

TABLE 4 Therapeutic effects of CNP gel preparation. Subject Subject 5Subject 1 Subject 2 Subject 3 Subject 4 (FIG. 1) Case Case 1 Case 6 Case5 Case 9 Case 10 Sex Female Female Male Female Male Age 21 years old 39years old 24 years old 9 months old 32 years old Dosage form CNP gel CNPgel CNP gel CNP gel CNP gel preparation preparation preparationpreparation preparation Dosage 30 μg/g 30 μg/g 30 μg/g 30 μg/g 30 μg/gNumber of Twice a day Twice a day Twice at 20-min Twice at 20-min 3times at 20- administration interval interval min interval Days of 4days 3 days Only 1 day when Only 1 day when Only 1 day whenadministration visiting the visiting the visiting the clinic. clinic.clinic. Application Face and neck Face and neck Back Forearm Upper limbregion Severity level Before: severe Before: severe Before: severeBefore: moderate Before: severe of rash by After: mild After: mildAfter: mild After: mild After: moderate Dermatological AssociationGuideline Severity level Erythema: 3 Erythema: 3 Erythema: 3 Erythema: 2Erythema: 3 evaluation of Edema/papulation: 3 Edema/papulation: 3Edema/papulation: 3 Edema/papulation: 2 Edema/papulation: 3 the rashOozing/crusting: 1 Oozing/crusting: 2 Oozing/crusting: 2Oozing/crusting: 2 Oozing/crusting: 3 region by Excoriation: 1Excoriation: 2 Excoriation: 2 Excoriation: 2 Excoriation: 3 SCORAD(before Lichenification: 2 Lichenification: 3 Lichenification: 1Lichenification: 1 Lichenification: 3 application) Dryness: 3 Dryness: 3Dryness: 2 Dryness: 2 Dryness: 3 Total: 13/18 Total: 16/18 Total: 13/18Total: 11/18 Total: 18/18 Severity level Erythema: 1 Erythema: 1Erythema: 1 Erythema: 1 Erythema: 1 evaluation of Edema/papulation: 1Edema/papulation: 0 Edema/papulation: 1 Edema/papulation: 1Edema/papulation: 1 the rash Oozing/crusting: 1 Oozing/crusting: 0Oozing/crusting: 1 Oozing/crusting: 1 Oozing/crusting: 0 region byExcoriation: 1 Excoriation: 0 Excoriation: 1 Excoriation: 0 Excoriation:1 SCORAD (after Lichenification: 0 Lichenification: 0 Lichenification: 0Lichenification: 0 Lichenification: 2 application) Dryness: 1 Dryness: 1Dryness: 1 Dryness: 1 Dryness: 2 Total: 5/18 Total: 2/18 Total: 5/18Total: 4/18 Total: 7/18 Detailed After 4 days of After 3 days of AfterAfter After description of application of application of application ofapplication of application of improvement 30-μg/g CNP gel 30-μg/g CNPgel 30-μg/g CNP gel 30-μg/g CNP gel 30-μg/g CNP gel status ofpreparation to preparation to preparation to preparation to preparationto symptoms both sides of both sides of left side of the the right theright upper the face and the face and back twice at forearm twice atlimb at 20-min neck twice a neck twice a 20-min interval, 20-mininterval, interval, day, erythema day, remarkable erythema with scalyerythema subjective was markedly effects were swelling on the andinfiltration burning reduced, scales shown and left side of the on theright sensation on the disappeared and erythema was back was cubitalfossa right side the skin texture almost markedly were reduced and(where CNP gel became fine and disappeared, in improved 45 min,erythema, preparation was softened. addition, scales to a mild papulesand applied) disappeared and symptom scales on the subsided, and theskin texture characterized by forearms were infiltration, became fine.mild erythema. improved. scales and erythema on the right side weremarkedly improved compared to those on the left side. Itching Before: 10Before: 10 Before: 10 (Infant) Before: 10 sensation After: 0 After: 1After: 2 After: 3 Non-recurrence 2 weeks 7 days 7 days 2 weeks or more 7days period

TABLE 5 Diagnosis prior to application of CNP aqueous-solutionpreparation. Subject Subject 6 Subject 7 (FIG. 2) (FIG. 3) Subject 8Subject 9 Subject 10 Case Case 2 Case 7 Case 8 Case 3 Case 4 Sex FemaleFemale Female Female Male Age 41 years old 29 years old 36 years old 38years old 32 years old Onset and Developed at 7 Developed in Developedin Developed at 2 He presents with course of years of age, infancy;symptoms infancy, having months after chronic recurrent disease havingrecurrent worsened since recurrent eczema birth; she dermatitis witheczema with about a half year with itching; she presents with itchingitching; in ago, showing presents with generalized-type repeatedly,which particular severe severe recurrent eczema extended to theinfiltrative infiltrative infiltration and with itching. whole bodysince erythema on the erythema on the erythema over the 2 years ago.face tends to four limbs and whole body; worsen acutely; face. withoutherpes virus application of infection on the “the strongest” face as asteroid external complication has drugs, exudate been repeated. leaksout. Family None Elder brother; None Mother; Atopic history Atopicdermatitis dermatitis Past Child asthma Allergic rhinitis Child asthmaBronchial asthma Allergic rhinitis history Scratch House dust: 3+ Housedust: 2+ House dust: 3+ House dust: 2+ House dust: 2+ test Mite: 3+Mite: 2+ Mite: 3+ Mite: 3+ Mite: 2+ Orchard grass: 2+ Cedar: 3+ Cedar:2+ Ragweed: 1+ Orchard grass: 3+ Orchard grass: 2+ Ragweed: 2+ Ragweed:1+ Diagnostic Infiltrative Infiltrative Infiltrative InfiltrativeInfiltrative findings erythema with erythema erythema with erythema,erythema, scales, itching lichenified itching lichenified and numerousassociated with lesions, associated with lesions, excoriations are sleeperythema, severe sleep erythema, severe observed over the disturbance,scales, adhesion disturbance, scales, adhesion whole body. lichenifiedof crusts, and lichenified of crusts, and lesions, and numerous lesions,and numerous erythema are excoriations are erythema are excoriations areobserved over the observed on the observed over the observed over thewhole body. On four limbs and whole body. whole body; she the face; sheface. is in the state presents with of erythroderma repeatedposteczematosa. exacerbation of infiltrative erythema with a strongburning sensation associated with capillary dilation. Symptoms of Rasheson the Rashes on the Rashes on the Rashes on the Rashes on theapplication face are mainly face are mainly face and neck are face aremainly forearms are regions characterized by characterized by mainlycharacterized by mainly erythema with erythema with characterized byerythema with characterized by severe swelling, severe severelichenification, erythema with infiltration or lichenification,infiltration, and as well as severe swelling, lichenification, as wellas erythema with papules, as well as as well as papules, edema.erosions, scales, scales, and papules, erosions erosions, scales andnumerous excoriations. and numerous and numerous excoriations.excoriations. excoriations. Effects of Not reduced by Although Steroidexternal Not reduced even Upon steroid steroid external infiltrativedrugs are not by “very strong” discontinuation external therapy.erythema on the effective for steroid external of drug face is reducederythema, therapy. steroid external by steroid infiltrative therapy, heis in external therapy, erythema and a state of it easily scales on theerythroderma with relapses and face and neck. appearance of tends toworsen. skin flush and desquamation over the whole body. Evaluation Mostsevere Severe Most severe Most severe Severe of symptoms

TABLE 6 Therapeutic effects of CNP aqueous-solution preparation. SubjectSubject 6 Subject 7 (FIG. 2) (FIG. 3) Subject 8 Subject 9 Subject 10Case Case 2 Case 7 Case 8 Case 3 Case 4 Sex Female Female Female FemaleMale Age 41 years old 29 years old 36 years old 38 years old 32 yearsold Dosage form CNP aqueous- CNP aqueous- CNP aqueous- CNP aqueous- CNPaqueous- solution solution solution solution solution preparationpreparation preparation preparation preparation Dosage 100 μg/ml 100μg/ml 100 μg/ml 100 μg/ml 100 μg/ml Number of Twice a day Twice a dayTwice a day Twice a day 3 times at 20-min administrations interval Daysof 4 days 4 days 10 days 4 days Only 1 day when administration visitingthe clinic. Application Face and neck Face Face and neck Face Noneregions Severity level Before: severe Before: severe Before: severeBefore: severe Before: severe of rash by After: mild After: mild After:moderate After: minor After: minor Dermatological Association GuidelineSeverity level Erythema: 3 Erythema: 3 Erythema: 3 Erythema: 3 Erythema:3 evaluation of Edema/papulation: 2 Edema/papulation: 3Edema/papulation: 3 Edema/papulation: 2 Edema/papulation: 2 the rashregion Oozing/crusting: 2 Oozing/crusting: 2 Oozing/crusting: 1Oozing/crusting: 2 Oozing/crusting: 2 by SCORAD Excoriation: 1Excoriation: 2 Excoriation: 2 Excoriation: 2 Excoriation: 2 (beforeLichenification: 3 Lichenification: 2 Lichenification: 3Lichenification: 2 Lichenification: 2 application) Dryness: 3 Dryness: 2Dryness: 3 Dryness: 3 Dryness: 2 Total: 14/18 Total: 14/18 Total: 15/18Total: 14/18 Total: 13/18 Severity level Erythema: 0 Erythema: 1Erythema: 1 Erythema: 1 Erythema: 0 evaluation of Edema/papulation: 0Edema/papulation: 0 Edema/papulation: 1 Edema/papulation: 0Edema/papulation: 1 the rash region Oozing/crusting: 0 Oozing/crusting:1 Oozing/crusting: 1 Oozing/crusting: 0 Oozing/crusting: 0 by SCORADExcoriation: 0 Excoriation: 1 Excoriation: 0 Excoriation: 1 Excoriation:0 (after Lichenification: 1 Lichenification: 0 Lichenification: 2Lichenification: 0 Lichenification: 0 application) Dryness: 1 Dryness: 1Dryness: 1 Dryness: 1 Dryness: 1 Total: 2/18 Total: 4/18 Total: 6/18Total: 3/18 Total: 2/18 Detailed After 4 days of On 4th day of After 3days of After 4 days of Sixty (60) min description of application ofapplication of application of application of after the improvement 100μg/ml CNP 100 μg/ml CNP 100-μg/ml CNP 100-μg/ml CNP application ofstatus of aqueous-solution aqueous-solution aqueous-solutionaqueous-solution 100 μg/ml CNP symptoms preparation to preparation, allpreparation to preparation, all aqueous-solution both sides of the ofthe both sides of the of the preparation 3 face and neck infiltrativeface and neck infiltrative times at 20 min twice a day, erythema, twicea day, erythema, interval, erythema was lichenified erythema andlichenified subjective markedly reduced lesions, infiltration werelesions, itching sensation and scales erythema, severe reduced anderythema, severe on the right disappeared, and scales, adhesion erosionswere scales, adhesion forearm was the skin texture of crusts, andalleviated. of crusts, and reduced and became fine. numerous numerouserythema was excoriations on excoriations were markedly the face weremarkedly improved, and markedly improved. inflammation and improved.dryness symptoms almost disappeared. Itching Before: 10 Before: 10Before: 10 Before: 10 Before: 10 sensation After: 1 After: 0 After: 4After: 0 After: 1 Non-recurrence 5 days 2 weeks or more 5 days 2 weeks 2weeks period

EXAMPLE 6

Details of the test examples of the CNP gel preparation and CNPaqueous-solution preparation summarized in Tables 3 to 6 are as follows.

CNP Gel Preparation, Test-Example 1 (Case 1; Subject 1; See Tables 3 and4):

Preliminary Test:

In the subject of Case 1, erythema, infiltrative erythema, and scaleswere observed on the face and neck. Steroid external drugs did not showany effects on these erythema, infiltrative erythema, and scales.

Therefore, the CNP gel preparation of the present invention was appliedonly to the right side of the face and neck of the subject, and a gelwithout CNP as the active ingredient was applied to the left side, twicea day, and symptoms were observed. The CNP gel preparation used was theCNP gel preparation (30 μg/g) obtained in Example 2.

Preliminary Test Results:

Ten min after the application, as a subjective symptom, a burningsensation subsided on the right side on which the CNP gel preparationwas applied, and 30 min later, erythema also began to become less severeto a certain degree. In addition, there were no irritation symptoms dueto the application of the CNP gel preparation.

Treatment and its Outcome:

From the above findings of the preliminary test, the CNP gel preparationof the present invention was judged to be effective in this case withoutside effects; therefore its application to both sides of the face andneck twice a day was started. As a result, erythema clearly improved bydays after the start of the treatment, while scales disappeared, and theskin was softened and its texture became finer. Furthermore, given thatthe itching sensation level before the application of the CNP gelpreparation is rated as 10, then the itching sensation level after theapplication was 0, i.e., it is completely disappeared. Here, evaluationof the itching sensation level was performed by the visual analoguescale method.

Clinical follow-up after this revealed that, when the application of theCNP gel preparation was discontinued for 4 or 5 days, recurrence ofminor erythema and scales was observed, but the symptoms did not worsenacutely, and the symptoms were improved without prolongation by theapplication of the CNP gel preparation for several days. It was alsoeffective for the exacerbation before the start of a menstrual period,and symptoms were improved to almost normal conditions with littleinflammatory symptom by the next morning, by the application twice a dayin the morning and evening.

CNP Gel Preparation, Test Example 2 (Case 6; Subject 2; See Tables 3 and4):

Treatment and its Outcome:

The subject of Case 6 presented with infiltrative erythema with itchingassociated with sleep disturbance, lichenified lesions, and erythemaover the whole body, and also infiltrative erythema with severe scalesand a strong burning sensation on the face. In addition, the subject wasin a state of erythroderma posteczematosa presumably due to thelong-term steroid external therapy, presenting with skin flush anddesquamation over the whole body. The severity level of the rash on theface was characterized mainly by severe infiltration, erythema andnumerous excoriations.

Under such conditions, using the right/left comparative method,initially, the CNP gel preparation with a concentration of 30 μg/gobtained in Example 2 was applied only to the right side of the face,and a gel alone was applied to the left side of the face. As a result,immediately after the application of the CNP gel preparation, a burningsensation on the right side subjectively subsided, and erythema began toimprove. Moreover, no symptoms of irritation due to the application ofthe CNP gel preparation were observed.

Subsequently, the CNP gel preparation with a concentration of 30 μg/gwas applied to both sides of the face and the neck twice a day. As aresult, significant effects were observed; erythema almost disappearedafter 3 days, and scales disappeared and the texture of the skin becamefiner.

The severity level of the rash on the face was improved by theapplication of the CNP gel preparation of the present invention fromsevere to mild, according to the classification based on theDermatological Association Guideline. Furthermore, by the application ofthe CNP gel preparation of the present invention, the itching sensationlevel was improved from 10 to 1 according to the visual analogue scalemethod.

Clinical follow-up after this revealed that, around 3 days after thediscontinuation of the present CNP gel preparation, mild erythemarecurred but did not worsen any further. In this case, when the presentCNP gel preparation was re-applied, these symptoms disappeared after 3or 4 days

CNP Gel Preparation, Test Example 3 (Case 5; Subject 3; See Tables 3 and4):

Treatment and its Outcome:

This test mainly aimed at evaluation of absorbability of the CNP gelpreparation at the dorsal region.

The subject of Case 5 had moderate to severe erythema, scales, andexcoriations over the whole body, and the severity level of the rash onthe back was characterized mainly by severe swelling, erythema withedema, and scales.

Under such condition, using the right/left comparative method, the CNPgel preparation with a concentration of 30 μg/g obtained in Example 2was applied only to the left side of the back of the subject twice at aninterval of 20 min, and a gel alone was applied to the right side of theback. As a result, erythema with swelling improved markedly on the leftside after 45 min, to a mild symptom characterized mainly by milderythema. The description in Table 4 shows the results of evaluation ofthe therapeutic effects of the CNP gel preparation at this point.

The above gel preparation (concentration of 30 μg/g) was applied to theface and neck of the same subject without right/left comparisons, twicea day for 3 days. As a result, erythema and scales on the face and neckmarkedly improved to be diagnosed as being a mild degree. Namely, theseverity level on the face and neck was improved from severe to mild,according to the classification based on the Dermatological AssociationGuideline.

These test results demonstrated that the CNP gel preparation of thepresent invention has excellent transdermal absorbability not only onthe face, neck and forearms, but also on the dorsal region.

CNP Gel Preparation, Test Example 4 (Case 9; Subject 4; See Tables 3 and4):

Treatment and its Outcome:

The subject of Case 9 was a female infant suffering from recurrenteczema with itching over the whole body, and erythema, papules, scalesand numerous excoriations were observed over the whole body. Inaddition, erythema, papules, scales and numerous excoriations wereobserved on both forearms.

Under such condition, the CNP gel preparation with a concentration of 30μg/g obtained in Example 2 was applied only to the right forearm of thesubject. The application was performed twice at an interval of 20 min.As a result, desquamative erythema and infiltration on the right cubitalfossa were reduced, and erythema, papules and scales on the forearm werealso improved.

Of particular note in this case is that improved condition wasmaintained for 2 weeks or longer after application only twice at thetime of visiting the clinic. The result of this test showed that scalesdisappeared, and skin texture became finer and softened, thus theeffectiveness in infant were also evident.

CNP Gel Preparation, Test Example 5 (Case 10; Subject 5; see Tables 3and 4):

Treatment and its Outcome:

The subject of Case 10 developed atopic dermatitis in infancy; sufferingfrom recurrent eczema with itching; presenting with infiltrativeerythema with itching associated with sleep disturbance, lichenifiedlesions and scales over the whole body; being associated chillysensation; and the subject was diagnosed as erythroderma posteczematosa.Particularly on the upper limbs, infiltrative erythema with a strongburning sensation was observed, signifying its intractable nature. Therash on both upper limbs was mainly characterized by severe swelling,infiltration, erythema and erosions, with numerous excoriations.

To this subject, using the right/left comparative method, the CNP gelpreparation with a concentration of 30 μg/g obtained in Example 2 wasapplied only to the right upper limb at an interval of 20 min, and a gelalone was applied to the left upper limb. As a result, the burningsensation on the right side, where the CNP gel preparation was applied,subsided after 60 min as a subjective symptom, and the infiltration,scales and erythema on the right side were markedly improved compared tothe right arm prior to the application, and compared to the left armwhere the gel preparation without CNP was applied three times at 20 mininterval. FIG. 1 is a photograph showing the result of application ofthe CNP gel preparation of the present invention to the subject.

CNP Aqueous-Solution Preparation, Test Example 1 (Case 2; Subject 6; seeTables 5 and 6):

Preliminary Test:

In the subject of Case 2, the rash on the face was characterized mainlyby erythema with severe swelling, infiltration and lichenification,papules, erosions, and numerous excoriations. The subject had a burningsensation as a subjective symptom. In addition, the subject had ahistory of recurring herpes virus infections, thus this is a case forwhich tacrolimus external therapy cannot be used.

As in the case of the test example 1, the CNP aqueous-solutionpreparation with a concentration of 100 μg/ml obtained in Example 3 wasapplied only to the right side of the face of the subject, and salinealone was applied to the left side.

Preliminary Test Results:

Ten min after the application, the burning sensation subsided on theright side of the face on which the CNP aqueous-solution preparation hadbeen applied, and erythema on the right side of the face also began tobe slightly improved 30 min later. In addition, irritation symptomsattributed to the application of the CNP aqueous-solution preparationwere completely absent. Since the subject felt that the burningsensation and heaviness on the skin were reduced and that thepreparation permeated into the skin, the preparation of the presentinvention was considered to be effective for reducing subjectivesymptoms. Moreover, apparent improvement in infiltration and erythemawas observed.

Treatment and its Outcome:

From the findings of the preliminary test above, the CNPaqueous-solution preparation of the present invention was judged to beeffective without side effects in this case; therefore, its applicationto both sides of the face and neck twice a day was initiated. As aresult, erythema was clearly improved after 4 days, while the scalesdisappeared and the texture of the skin became finer. Furthermore, whilethe itching sensation level before application of the CNPaqueous-solution preparation was scored 10 based on the visual analoguescale method, it was markedly improved to 1 after the application.

Clinical follow-up after this revealed that, when the application of theCNP aqueous-solution preparation was discontinued for 4 or 5 days, milderythema recurred, but the symptoms did not worsen any further. Inaddition, this recurrence of mild erythema was improved again by furtherapplication of the CNP aqueous-solution preparation for a period of 3-4days. FIG. 2 is a photograph showing the results of application of theCNP aqueous-solution preparation of the present invention to thesubject.

Moreover, the symptoms of this subject were markedly improved bycontinuous application of the CNP aqueous-solution preparation;accordingly, the subject became able to wear makeup. Furthermore, thesafety and efficacy of the present preparation as an externallyapplicable preparation to the patients with herpes virus infectioncomplications, to whom tacrolimus external therapy cannot be applied,has been demonstrated by this test.

Dosage-Finding Study:

A dose-finding study was performed with the same subject. With the CNPsolution preparation with a CNP concentration of 50 μg/ml, the healingprocess of the skin was slightly slower compared to that with theconcentration of 100 μg/ml, and the treated skin was slightly rough tothe touch. Although the CNP aqueous-solution preparation with a CNPconcentration of 200 μg/ml remained to cause no irritation, this did notdouble its effectiveness.

CNP Aqueous-Solution Preparation, Test Example 2 (Case 7; Subject 7; seeTables 5 and 6):

Treatment and its Outcome:

The subject of Case 7 presented with infiltrative erythema, lichenifiedlesions, erythema, severe scales, adhesion of crusts and numerousexcoriations on the four limbs and face, and the rash on the face wascharacterized mainly by erythema with lichenification, papules,erosions, scales and numerous excoriations.

Under such conditions, using the right/left comparative method,initially, the CNP aqueous-solution preparation with a concentration of100 μg/ml obtained in Example 3 was applied only to the right side ofthe face, and saline alone was applied to the left side of the face.

As a result, 20 min after application, a the subjective symptom ofburning sensation on the right side of the face subsided, and erythemabegan to be slightly improved. No irritation symptom attributable to theapplication of the CNP aqueous-solution preparation was observed at all.On the 4th day from the start of the application of the CNPaqueous-solution preparation, all of the infiltrative erythema,lichenified lesions, erythema, severe scales, adhesion of crusts andnumerous excoriations on the face were markedly improved. By theapplication of the CNP aqueous-solution preparation of the presentinvention, the itching sensation level was improved from 10 to 0, theabsence of subjective itching, based on the visual analogue scalemethod. In addition, the severity level of the rash on the face wasimproved from severe to mild by the application of the CNPaqueous-solution preparation, in accordance with the classification bythe Dermatological Association Guideline. Subsequently, follow-up showedno recurrence of erythema even after discontinuation of theadministration of the CNP aqueous-solution preparation. FIG. 3 is aphotograph showing the application of the CNP aqueous-solutionpreparation of the present invention to the subject.

CNP Aqueous-Solution Preparation, Test Example 3 (Case 8; Subject 8; seeTables 5 and 6):

Treatment and its Outcome:

The subject of Case 8 presented with infiltrative erythema with itchingassociated with sleep disturbance, lichenified lesions, and erythemaover the whole body; had severe infiltration and erythema due torecurrence of eczema with itching over the whole body, and was in astate in which exudate would come out if not treated by the applicationof “the strongest” steroid external drugs.

Under such conditions, we attempted to apply the CNP aqueous-solutionpreparation with a concentration of 100 μg/ml obtained in Example 3 tothe erythema, infiltrative erythema and scales on the face and neck, theareas in which no therapeutic effect was observed with steroid externaldrugs. The number of application was twice a day.

Using the right/left comparative method, the CNP aqueous-solutionpreparation was first applied to the right side of the face and necktwice at 20 min interval, and saline alone was applied to the left side.As a result, infiltration and erythema began to be slightly improved onthe right side. No irritation symptom attributable to the CNPaqueous-solution preparation was observed at all.

Subsequently, the CNP aqueous-solution preparation was applied to bothsides of the face and neck twice a day. As a result, erythema andinfiltration were improved and erosions were also reduced after 3 days.The application of the CNP aqueous-solution preparation reduced itching,and the itching sensation level was improved from 10 to 4 based on thevisual analogue scale method. The severity level of the rash 3 daysafter the start of the application was improved from most severe tomoderate by the standards of the Guideline 2005. Subsequent furthercontinuous application of the CNP aqueous-solution preparation twice aday for 7 days further improved the rash, and moderate infiltrativeerythema was improved to mild erythema.

CNP Aqueous-Solution Preparation, Test Example 4 (Case 3; Subject 9; seeTables 5 and 6):

Treatment and its Outcome:

The subject of Case 3 presented with infiltrative erythema, lichenifiedlesions, erythema, severe scales, adhesion of crusts, and numerousexcoriations over the whole body, and the rash on the face wascharacterized mainly by erythema with lichenification, papules,erosions, scales, and numerous excoriations.

The CNP aqueous-solution preparation with a concentration of 100 μg/mlwas applied to both sides of the face of the subject. As a result, thesubjective symptom of sensation of tight-stretched skin was improvedafter 10 min. Then erythema was improved and the skin became soft to thetouch with a finer texture from after 30 min. No irritation symptomswere observed with the external application, and the efficacy forepithelization of erosion and excoriation also became evident.Furthermore, findings about some beneficial changes such as reduction ofscales and becoming finer skin texture were obtained. After 4 days fromthe application of the CNP aqueous-solution preparation of 100 μg/ml,all of the infiltrative erythema, lichenified lesions, erythema, severescales, adhesion of crusts, and numerous excoriations were markedlyimproved. While the itching sensation level before application was ratedas 10, subjective itching completely disappeared after the applicationto a level of 0.

Regarding the severity of erythema on the face after application of theCNP aqueous-solution preparation, there were a little inflammatorysymptoms with almost no dryness, and the severity level of the rashbased on the Dermatological Association Guideline was markedly improvedfrom severe to minor.

From the above findings, the CNP aqueous-solution preparation wasconcluded to be very effective in this case. Subsequent follow-up showedno recurrence of erythema after discontinuation of the application ofthe present preparation.

CNP Aqueous-Solution Preparation, Test Example 5 (Case 4; Subject 10;see Tables 5 and 6):

Treatment and its Outcome:

This test mainly aimed at evaluation of absorbability of the CNPpreparations of the present invention on the forearms, on whichabsorbability is presumed to be inferior to that on the face.

The subject of Case 4 presented with moderate erythema, scales andexcoriation over the whole body, and the rash on the forearms wascharacterized mainly by erythema with severe swelling, scales, andexcoriations.

Under such conditions, 100 μl of the CNP aqueous-solution preparationwith a concentration of 100 μg/ml was applied to the right forearm 3times at 20 min interval, and saline alone was applied to the leftforearm. As a result, 60 min later, subjective itching was reduced onthe right forearm to which the CNP aqueous-solution preparation wasapplied; and erythema was also markedly improved; and inflammation anddryness symptoms mostly disappeared as well. No irritation symptomassociated with the application of the aqueous-solution preparation wasobserved at all. Thus, the CNP aqueous-solution preparation of thepresent invention exhibited remarkable effects on the forearms of thesubject of this case as well. These findings indicated that thepreparation of the present invention is also effective to the regionsother than the face where the drug absorbability is presumed to berelatively high, namely the disorders on forearms. The rash on theforearms was improved by the administration of the CNP aqueous-solutionpreparation, from severe to minor level according to the classificationbased on the Dermatological Association Guideline. In addition,application of the CNP aqueous-solution preparation improved the itchingsensation level from 10 to 1 based on the visual analogue scale method.

Next, CNP gel-base preparations were tested. “Gel-base preparation”differs from “gel preparation” in that the former does not comprisedipotassium glycyrrhizinate, allantoin, pyridoxine hydrochloride,xanthan gum and vitamin E. The effects of CNP preparations would be moreclearly demonstrated to be due to the effects of CNP itself byconfirming the effects of CNP in a preparation without these components,which could have some efficacy potentially.

EXAMPLE 7

1. Production of CNP Gel-Base Preparation:

Preparation of the gel was performed as follows.

0.1 g of methyl parahydroxybenzoate (product name: Mekkins M, Ueno FineChemicals Industry), 0.2 g of phenoxyethanol, and 3.0 g of1,2-pentanediol were measured in the same container, dissolved at 60-70°C., and introduced into a mixing kettle. The, 6.0 g of concentratedglycerin was introduced and a mixture of 0.44 g of carboxy vinyl polymer(product name: Carbopol 940, Lubrisol Advanced Materials, Inc.) and 0.08g of xanthan gum (product name: Keltrol T, CP Kelco) were added, andstirred with a paddle at 15 rpm to be sufficiently dispersed. Next,while stirring with a paddle at 15 rpm, 83.95 g of purified water wasgradually introduced and the mixture was dissolved by stirring at akettle temperature of 70-80° C., with a paddle at 20 rpm and a disperserat 1500-2000 rpm. After stopping the disperser, dissolution wasconfirmed and cooling was immediately started; when the kettletemperature approached around 40° C., 6.0 g of Lubrajel NP from ISPJapan, Ltd. (2.7 g of Glycerin, 0.06 g of carboxy vinyl polymer, 0.018 gof sodium polyacrylate, 3.222 g of water) was added and mixedhomogeneously with a paddle at 20 rpm, then 0.230 g of potassiumhydroxide was added for neutralization, and when the kettle temperaturereached 25° C., the rotation of the paddle was terminated to obtain agel-base.

Then, 3 mg of human CNP-22 (Peptide Institute, Inc.) as a principalagent was dissolved in 3 ml of saline to obtain the CNP solution with aconcentration of 1000 μg/ml, and 1 ml of this CNP solution washomogeneously stirred and mixed in 19 g of the gel-base obtained asabove, to produce the gel-base preparation with a concentration of 50μg/g.

Similarly, 600 μl of the above CNP solution with a concentration of 1000μg/ml was diluted with 400 μl of saline to prepare the concentration of600 μg/ml, then the 1 ml of resulting solution was homogeneously stirredand mixed in 19 g of the gel-base obtained as above, to produce thegel-base preparation with a concentration of 30 μg/g.

EXAMPLE 8

2. Diagnosis of Subjects:

Prior to administration of the CNP gel-base preparation of the presentinvention, history taking from the subjects, scratch tests for allergensand a diagnosis were conducted. Table 7 (subjects 11-15) and Table 9(subjects 16-20) show the results of the subjects′ history taking anddiagnosis, i.e., sex, age, onset and disease course, family history,past history, scratch test results, diagnostic findings, and symptomevaluations based on “Guideline 2005” of the subject in each case.

EXAMPLE 9

3. Therapeutic Effects on Subjects:

Therapeutic effects of the CNP gel-base preparation of the presentinvention are shown in Table 8 (subjects 11-15) and Table 10 (subjects16-20). In Tables 8 and 10, “itching sensation” represents a comparisonof the itching sensation evaluated using the visual analogue scalemethod before and after treatment. Similarly, “non-recurrence period”refers to the period after discontinuation of the treatment by thepreparation of the present invention subsequent to the improvement ofsymptoms, in which relapse of the symptoms did not occur. In order toevaluate objectively, photographs were taken before and afterapplication of CNP preparations for all cases. Of these, photographs ofsome cases are shown in the figures.

As shown in Tables 7 to 10, simply applying the CNP gel-basepreparations of the present invention to affected areas of atopicdermatitis twice a day for 2-4 days, resulted in itching sensation to bemostly disappeared, and the severity levels of the rash in terms ofexternal appearance was markedly improved in accordance with theDermatological Association Guideline and with the SCORAD method.

The CNP gel-base preparations of the present invention are almostidentically effective with both concentrations of 30 μg/g and 50 μg/g,and the preparations showed their efficacy regardless of sex over abroad range of ages from 21 to 39 years old. In addition, thepreparations were effective for any regions including the face, neck andupper limbs. Furthermore, atopic dermatitis was improved in the patientswho have immunoreactivity for a wide range of allergens, from patientswith immunoreactivity to house dust and mites, to patients withimmunoreactivity for all of house dust, mites, cedar, orchard grass, andragweed.

The CNP gel-base preparations of the present invention markedly improvedatopic dermatitis of patients with a familial allergic diathesis. Inaddition, the CNP gel-base preparations of the invention markedlyimproved the symptoms of atopic dermatitis in subjects 11-18, who haddeveloped the disease in their infancy and had recurrence repeatedly.Additionally, the CNP gel-base preparations of the present inventionmarkedly improved the symptoms of intractable, frequently-recurrentatopic dermatitis. Moreover, it is surprising that the CNP gel-basepreparations of the present invention markedly improved symptoms ofatopic dermatitis which steroid external therapy had failed toalleviate. Furthermore, it deserves a special note that the recurrenceof atopic dermatitis was not observed for at least 5 days, and forlonger cases 2 weeks or more, after discontinuation of the applicationof the CNP gel-base preparations of the invention.

Here, the above therapeutic effects on atopic dermatitis were moreclearly demonstrated to be attributable to the efficacy of CNP itselfsince the CNP gel-base preparation of the present invention differs from“gel preparation” in that the former does not comprise dipotassiumglycyrrhizinate, allantoin, pyridoxine hydrochloride, xanthan gum, andvitamin E.

TABLE 7 Diagnosis prior to application of CNP gel-base preparation.Subject Subject 11 Subject 12 Subject 13 Subject 14 Subject 15 Case — —— — — Sex Male Female Female Female Male Age 31 years old 23 years old39 years old 39 years old 21 years old Onset and Developed in Developedin Developed on the Developed in Developed at the course of infancy,having infancy, having face in infancy; infancy, having age of 4, havingdisease recurrent eczema recurrent eczema since around 20 recurrenteczema recurrent eczema with itching; with itching; years of age, withitching; in with itching; infiltrative eczema also dry eczema particularrecently, erythema on the developed and extended to infiltrativeerythema on the face appeared frequently almost whole erythema on theface and neck since he started recurred on the body including neckinduces began to show to work, and it face starting the face. The strongitching, intractable has recurred several years face in and the regionnature. frequently. ago. particular has is always repeatedly wrapped inbecome red with bandages, with fever as if it complication of gotburned, and intractable has also been atopic alopecia. severely dry formany years. Family Child; Atopic Mother; Atopic Mother; Atopic Father;Atopic Mother and history dermatitis dermatitis dermatitis dermatitisgrandmother; Atopic dermatitis Past Allergic Allergic Bronchial asthmaAllergic history rhinitis rhinitis, rhinitis Bronchial asthma ScratchHouse dust: 2+ House dust: 2+ House dust: 2+ House dust: 2+ House dust:2+ tests Mite: 3+ Mite: 3+ Mite: 2+ Mite: 3+ Mite: 3+ Cedar: 2+ Cedar:1+ Cedar: 1+ Cedar: 2+ Cedar: 2+ Orchard grass: 2+ Orchard grass: 3+Orchard grass: 2+ Orchard grass: 1+ Ragweed: 1+ Ragweed: 1+ Ragweed: 1+Diagnostic Erythema with Up to moderate Infiltrative Erythema withInfiltrative findings severe swelling/ erythema, erythema,lichenification, erythema, edema/ scales, a small lichenified severescales, erythema, infiltration/ number of lesions, adhesion of scales,and lichenification, papules, and erythema are crusts, andlichenification as well as many excoriations are observed on theerosions are are observed on papules, severe observed on the face, neckand observed on the the face and scales, adhesion face. back, face,neck, four neck. of crusts, accompanied by limbs and body vesicles, andstrong itching trunk. erosions are with sleep observed on thedisturbance.. face. Symptoms of Rash on the face Rash on the face Rashon the face Rash on the face Rash on the face application are associatedis mainly is mainly and neck are and neck is regions with erythemacharacterized by characterized by associated with erythema with withsevere erythema, edema, erythema with erythema with lichenification/swelling/edema/ papules, lichenification, infiltration or infiltration.infiltration/ or erosions, and and scales. lichenification,lichenification, excoriations. as well as adhesion of severe scales,crusts and and adhesion of vesicles. crusts. Effects of Relapsed on Noimprovement Relapsed on Relapsed on Relapsed on steroid discontinuationeven by “very discontinuation discontinuation discontinuation externalof steroid strong” steroid of steroid of steroid of steroid drugexternal therapy external external external external therapy. therapy;in therapy. therapy. particular intractable nature is shown on the face.Evaluation Severe Moderate Severe Severe Severe of symptoms

TABLE 8 Therapeutic effects of CNP gel-base preparation. Subject Subject11 Subject 12 Subject 13 Subject 14 Subject 15 Case — — — — — Sex MaleFemale Female Female Male Age 31 years old 23 years old 39 years old 39years old 21 years old Dosage form CNP gel-base CNP gel-base CNPgel-base CNP gel-base CNP gel-base preparation preparation preparationpreparation preparation Dosage 30 μg/g 30 μg/g 30 μg/g 30 μg/g 30 μg/gNumber of Twice a day Twice a day Twice a day Twice a day Twice a dayadministration Days of 3 days 3 days 3 days 3 days 3 days administrationApplied region Face Face Face Face and neck Face and neck Severity levelBefore: severe Before: severe Before: severe Before: severe Before:severe of rash by After: mild After: minor After: mild After: mildAfter: mild Dermatological Association Guideline Severity levelErythema: 3 Erythema: 3 Erythema: 3 Erythema: 2 Erythema: 2 evaluationof Edema/papulation: 3 Edema/papulation: 2 Edema/papulation: 2Edema/papulation: 2 Edema/papulation: 2 the rash Oozing/crusting: 2Oozing/crusting: 2 Oozing/crusting: 1 Oozing/crusting: 2Oozing/crusting: 1 region by Excoriation: 1 Excoriation: 2 Excoriation:1 Excoriation: 2 Excoriation: 1 SCORAD (before Lichenification: 2Lichenification: 1 Lichenification: 3 Lichenification: 2Lichenification: 2 application) Dryness: 2 Dryness: 2 Dryness: 3Dryness: 3 Dryness: 2 Total: 13/18 Total: 12/18 Total: 13/18 Total:13/18 Total: 10/18 Severity level Erythema: 1 Erythema: 0 Erythema: 1Erythema: 1 Erythema: 1 evaluation of Edema/papulation: 0Edema/papulation: 0 Edema/papulation: 0 Edema/papulation: 0Edema/papulation: 0 the rash Oozing/crusting: 1 Oozing/crusting: 0Oozing/crusting: 0 Oozing/crusting: 1 Oozing/crusting: 0 region byExcoriation: 0 Excoriation: 1 Excoriation: 0 Excoriation: 1 Excoriation:0 SCORAD (after Lichenification: 0 Lichenification: 0 Lichenification: 0Lichenification: 0 Lichenification: 0 application) Dryness: 1 Dryness: 1Dryness: 1 Dryness: 1 Dryness: 1 Total: 3/18 Total: 2/18 Total: 2/18Total: 4/18 Total: 2/18 Detailed After 3 days of After 3 days of By theAfter 3 days of After 3 days of description of application ofapplication of 30 μg/g application of 30 μg/g application of applicationof improvement 30-μg/g CNP gel- CNP gel-base CNP gel-base 30-μg/g CNPgel- 30-μg/g CNP gel- status of base preparation preparation topreparation to base preparation base preparation symptoms to the facetwice the face twice a the face twice a to the face twice to the faceand a day, erythema, day, erythema, day, after 2 days a day, erythema,neck twice a day, infiltration and infiltration, erythema wasinfiltration, erythema, itching markedly scales and reduced, and scalesand infiltration, and improved and the itching were infiltration anditching were itching were skin textures markedly itching were markedlymarkedly became fine. improved. markedly improved. improved. improved,and burning sensation disappearedon day 3. Itching Before: 10 Before: 10Before: 10 Before: 10 Before: 10 sensation After: 0 After: 0 After: 1After: 0 After: 0 Non-recurrence 2 weeks 2 weeks 2 weeks or more 5 days2 weeks period

TABLE 9 Diagnosis prior to application of CNP gel-base preparation.Subject Subject 17 Subject 20 Subject 16 (FIG. 4) Subject 18 Subject 19(FIG. 5) Case — — — — — Sex Male Female Male Female Female Age 25 yearsold 36 years old 22 years old 31 years old 35 years old Onset and Hadrecurrent Developed in Developed in She had eczema Recurrent course ofeczema with infancy, and infancy; at cubital erythema disease itchingsince symptoms worsen symptoms were fossae and appeared since infancy,and due to change of initially only popliteal fossae 21 years of agesymptoms seasons and on the body that worsened by when she beganworsened due to sweating. trunk, but sweating, and to work a nightpsychological Application of rapidly worsened which was shift, andstress during various external to extend to reduced extended to thethese last preparations did almost the whole temporarily by whole body.several years; not stop the body including steroid external by theinflammation at the face. In application but application of all, andsleep particular, relapsed steroid external disturbance due symptomsworsen repeatedly. drugs every day, to itching when he is in Sincearound skin flush, continued; dusty 2002 when she scales and severeswelling conditions. started to work, infiltrative prohibits erythemaerythema became bending of the appeared on the observed over arms. facedue to the whole body. leisureless life and stress caused byinterpersonal relationships. Family Father; Atopic Father; AllergicMother; Atopic Mother; Elder brother; history dermatitis rhinitis,dermatitis Bronchial asthma Bronchial asthma conjunctivitis PastAllergic Allergic Allergic Allergic Allergic history rhinitis rhinitisrhinitis, rhinitis, rhinitis, Bronchial asthma ConjunctivitisConjunctivitis Scratch House dust: 3+ House dust: 2+ House dust: 2+House dust: 3+ House dust: 3+ test Mite: 3+ Mite: 3+ Mite: 3+ Mite: 3+Mite: 3+ Cedar: 2+ Cedar: 2+ Cedar: 3+ Cedar: 2+ Orchard grass: 3+Orchard grass: 1+ Ragweed: 1+ Ragweed: 2+ Diagnostic InfiltrativeErythema with Infiltrative Infiltrative Infiltrative findings erythema,lichenification, erythema with erythema, erythema, lichenifiedinfiltrative strong itching lichenified lichenified lesions, erythema,severe associated with lesions, lesions, erythema, severe scales,adhesion sleep erythema, and erythema, severe scales, adhesion ofcrusts, disturbance, numerous scales, adhesion of crusts, and vesicles,and erythema, and excoriations are of crusts and numerous erosions areexcoriations are observed over numerous excoriations are observed overobserved on the whole body. excoriations are observed over the wholebody. almost whole Have strong observed over the whole body. bodyincluding itching the whole body. He is in a state face and neck.associated with of erythroderma sleep posteczematosa. disturbance.Symptoms of Rash on the face Rash on the Rash on the face Rash on theface Rash on the application and neck face, neck and is mainly and neckupper limbs are regions includes four limbs are characterized byconsists of associated with erythema with associated with erythema withsevere swelling/ severe swelling/ severe swelling/ erythema withlichenification, edema/ edema/ infiltration/ or infiltration orerosions, infiltration/ infiltration/ lichenification. lichenification,scales, and erythema, erythema. severe scales, numerous vesicles and andnumerous excoriations. erosions. excoriations. Effects of Skin flush andSymptoms are not Recurrence Skin flush and Skin flush and steroiddesquamation alleviated by occurred soon desquamation desquamationexternal appear over the steroid external after appear after appearafter drug whole body after therapy at all. discontinuationdiscontinuation discontinuation discontinuation of steroid of steroid ofsteroid of steroid external external external external therapy.application. application. application, resulting in a state oferythroderma. Evaluation Most severe Most severe Most severe Severe Mostsevere of symptoms

TABLE 10 Therapeutic effects of CNP gel-base preparation. SubjectSubject 17 Subject 20 Subject 16 (FIG. 4) Subject 18 Subject 19 (FIG. 5)Case — — — — — Sex Male Female Male Female Female Age 25 years old 36years old 22 years old 31 years old 35 years old Dosage form CNPgel-base CNP gel-base CNP gel-base CNP gel-base CNP gel-base preparationpreparation preparation preparation preparation Dosage 30 μg/g 30 μg/g50 μg/g 50 μg/g 50 μg/g Number of Twice a day Twice a day Twice a dayTwice a day Twice a day administration Days of 3 days 2 days 3 days 2days 4 days administration Applied region Face and neck Face, neck andFace Face and neck Forearms upper limbs Severity level Before: severeBefore: severe Before: severe Before: severe Before: severe of rash byAfter: mild After: mild- After: mild After: mild After: minorDermatological moderate Association Guideline Severity level Erythema: 3Erythema: 3 Erythema: 3 Erythema: 3 Erythema: 3 evaluation ofEdema/papulation: 3 Edema/papulation: 2 Edema/papulation: 2Edema/papulation: 3 Edema/papulation: 3 the rash Oozing/crusting: 3Oozing/crusting: 3 Oozing/crusting: 2 Oozing/crusting: 3Oozing/crusting: 1 region by Excoriation: 2 Excoriation: 3 Excoriation:1 Excoriation: 2 Excoriation: 1 SCORAD (before Lichenification: 3Lichenification: 3 Lichenification: 3 Lichenification: 2Lichenification: 1 application) Dryness: 3 Dryness: 3 Dryness: 3Dryness: 3 Dryness: 3 Total: 17/18 Total: 17/18 Total: 14/18 Total:16/18 Total: 12/18 Severity level Erythema: 1 Erythema: 1 Erythema: 1Erythema: 1 Erythema: 0 evaluation of Edema/papulation: 1Edema/papulation: 1 Edema/papulation: 1 Edema/papulation: 1Edema/papulation: 0 the rash Oozing/crusting: 0 Oozing/crusting: 1Oozing/crusting: 1 Oozing/crusting: 1 Oozing/crusting: 0 region byExcoriation: 1 Excoriation: 2 Excoriation: 1 Excoriation: 0 Excoriation:0 SCORAD (after Lichenification: 1 Lichenification: 1 Lichenification: 1Lichenification: 0 Lichenification: 0 application) Dryness: 1 Dryness: 1Dryness: 2 Dryness: 1 Dryness: 1 Total: 5/18 Total: 7/18 Total: 7/18Total: 4/18 Total: 1/18 Detailed After 2 or 3 days After 2 days of Afterapplication After 2 days of After 3 days of description of ofapplication of application of 30 μg/g of 50 μg/g CNP application of 50μg/g application of 50 μg/g improvement 30 μg/g CNP gel- CNP gel-basegel-base CNP gel-base CNP gel-base status of base preparationpreparation to preparation twice preparation to preparation twicesymptoms to the face and the face, neck a day, dryness the face and necka day, erythema neck twice a day, and upper limbs and itching were twicea day, was markedly tingling twice a day, markedly improved erythema wasreduced, and the sensation erythema, on the next day. reduced, and skintexture disappeared, and infiltration, 2 days later, itching became fineon erythema, scales and erythema was disappeared. the 4thday.infiltration and itching were reduced, and only Slight dryness itchingwere markedly slight scales and erythema markedly improved, and sheremained on the remained. improved. was able to 3rd day. sleep. ItchingBefore: 10 Before: 10 Before: 10 Before: 10 Before: 10 sensation After:2 After: 2 After: 3 After: 0 After: 0 Non-recurrence 7 days 5 days 5days 5 days 7 days period

EXAMPLE 10

1. Production of CNP Ointment Preparation:

Ointments were prepared as follows: 3 mg of human CNP-22 (PeptideInstitute, Inc.) as a principal agent was dissolved in 3 ml of saline toobtain the CNP solution with a concentration of 1000 μg/ml; 1 ml of thisCNP solution was homogeneously mixed in 9 g of white vaseline ofJapanese Pharmacopoeia by high-speed stirring, to adjust itsconcentration to 100 μg/g.

Similarly, 500 μl of the above CNP solution with a concentration of 1000μg/ml was diluted with 500 μl of saline to adjust its concentration at500 μg/ml, and 1 ml of this solution was homogeneously mixed in 9 g ofwhite vaseline of the Japanese Pharmacopoeia by high-speed stirring, toadjust its concentration at 50 μg/g.

In addition, 300 μl of the above CNP solution with a concentration of1000 μg/ml was diluted with 700 μl of saline to adjust its theconcentration at 300 μg/ml, and 1 ml of this solution was homogeneouslymixed in 9 g of white vaseline of the Japanese Pharmacopoeia byhigh-speed stirring, to adjust its concentration to 30 μg/g.

EXAMPLE 11

2. Diagnosis of Subjects:

Prior to administration of the CNP ointment preparation of the presentinvention, history taking from subjects, scratch tests for allergens anddiagnosis were conducted. Table 11 (subjects 21-25), Table 13 (subjects26-30), and Table 15 (subjects 31-35) show the results of the subjects′history taking and diagnosis, i.e., sex, age, onset and course ofdisease, family history, past history, scratch test results, diagnosticfindings, and symptom evaluation based on “Guideline 2005” of thesubject in each case.

EXAMPLE 12

3. Therapeutic Effect on Subjects:

Therapeutic effects of the CNP ointment preparation of the presentinvention are shown in Table 12 (subjects 21-25), Table 14 (subjects26-30), and Table 16 (subjects 31-35). In Tables 12, 14 and 16, “itchingsensation” represents a comparison of the itching sensation evaluatedusing the visual analogue scale method before and after treatment.Similarly, the “non-recurrence period” refers to the period afterdiscontinuation of the treatment by the preparation of the presentinvention subsequent to the improvement of symptoms, for which relapseof the symptoms did not occur.

As shown in Tables 11 through 16, simply applying the CNP ointmentpreparation of the present invention to affected areas of atopicdermatitis twice a day for 2-3 days resulted in the itching sensation todisappear mostly, and the severity levels of the rash in terms ofexternal appearance was markedly improved in accordance with theDermatological Association Guideline or in terms of external appearanceaccording to the SCORAD method. Here, in order to evaluate objectively,photographs were taken before and after the application of CNPpreparations for all cases. Of these, photographs of some of the casesare shown in the figures.

The CNP ointment preparations of the present invention are almostidentically effective with concentrations of 30 μg/g, 50 μg/g, and 100μg/g, and the preparations showed their efficacy regardless of sex overa broad range of ages from 3 to 56 years old. In addition, thepreparations were effective for any regions including the face, neck,back, and upper limbs. Furthermore, atopic dermatitis was improved inthe patients who have immunoreactivity for a wide range of allergens,from patients with immunoreactivity to house dust, mites, and orchardgrass to patients with immunoreactivity to all of house dust, mites,cedar, orchard grass, and ragweed.

The CNP ointment preparations of the present invention markedly improvedatopic dermatitis of the patients with a familial allergic diathesis. Inaddition, the CNP ointment preparations of the present inventionmarkedly improved symptoms of atopic dermatitis in the subjects who haddeveloped the disease in their infancy and had recurrence repeatedly.Additionally, the CNP ointment preparations of the present inventionalso markedly improved symptoms of intractable, frequently-recurrentatopic dermatitis. Moreover, it is surprising that the CNP ointmentpreparations of the present invention markedly improved symptoms ofatopic dermatitis steroid external therapy had failed to alleviate.Furthermore, it deserves special note that recurrence of atopicdermatitis was not observed for at least 5 days, and for longer cases, 2weeks or more, after discontinuation of the application of the CNPointment preparations of the present invention.

Here, since the CNP ointment preparations of the present invention donot comprise any components, other than CNP, which could potentiallyhave some efficacy, the above therapeutic effects on atopic dermatitisare clearly demonstrated to be the efficacy of the CNP itself.

TABLE 11 Diagnosis prior to application of CNP ointment preparation.Subject Subject 21 Subject 23 (FIG. 6) Subject 22 (FIG. 7) Subject 24Subject 25 Case — — — — — Sex Female Male Female Female Male Age 21years old 21 years old 28 years old 33 years old 39 years old Onset andDeveloped in Developed at 3 Developed at Developed in Eczema appearedcourse of infancy; months of age the time of infancy, has on the facedisease symptoms worsen with eczema being a junior recurrent eczemasince around 17 in dry seasons, mainly on the high school with itching;years of age, erythema with four limbs; student with from around and heitching appears asthma worsened symptoms on the starting to repeatedlymainly on the 4 years ago, and face; eczema work, suffers from dry face,neck, dermatitis with itching infiltrative dermatitis that upper limbsand rapidly expanded which worsens erythema worsens in body trunk, anddue to the with sweating appeared winter. the symptoms stress of haveappeared particularly on Thereafter he recurred examinations andrepeatedly on the face and has continuously repeatedly family thecubital neck, which visited a despite the relationships fossae and back,extended to the dermatologist application of starting 3 years etc.;since 3 whole body and for treatment, steroid external ago. years ago,she claims that but symptoms drugs. erythema has she can hardly showintractable always appeared sleep because of nature even despite steroiditching. against long- external term steroid application. externaltherapy. Family Mother; Atopic Younger sister, Elder brother; Mother andChild; Atopic history dermatitis Father; Atopic Atopic younger brother;dermatitis dermatitis dermatitis, Atopic Mother: allergic Allergicdermatitis rhinitis rhinitis Past Allergic Bronchial asthma AllergicAllergic Bronchial history rhinitis, Atopic rhinitis, rhinitis, asthma,Allergic conjunctivitis Bronchial asthma Conjunctivitis rhinitis ScratchHouse dust: 1+ House dust: 2+ House dust: 2+ House dust: 2+ House dust:3+ test Mite: 1+ Mite: 3+ Mite: 2+ Mite: 2+ Mite: 3+ Cedar: 2+ Cedar: 2+Cedar: 2+ Cedar: 1+ Cedar: 2+ Orchard grass: 2+ Orchard grass: 2+Orchard grass: 3+ Orchard grass: 1+ Orchard grass: 2+ Ragweed: 2+Ragweed: 2+ Ragweed: 1+ Diagnostic Infiltrative InfiltrativeInfiltrative Infiltrative Infiltrative findings erythema, erythema,erythema with erythema with erythema with erythema, severe erythema,severe strong itching strong itching strong itching scales, adhesionscales, adhesion associated with and erythema are associated with ofcrusts, and of crusts, and sleep observed on the sleep numerous edemaare disturbance, face, neck and disturbance, excoriations are observedon erythema, and body trunk. lichenified observed over almost wholeexcoriations are lesions, and the whole body, body, observed on theerythema are and particularly accompanied by face, neck, four observedover severe on the hair loss. limbs and back. the whole body. face andneck. Symptoms of Rash on the face Rash on the face Rash on the faceRash on the face Rash on the face application and neck is and neck isand neck is and neck is and neck is regions mainly mainly mainly mainlymainly characterized by characterized by characterized by characterizedby characterized by erythema with erythema with severe erythema witherythema with infiltration, infiltration and infiltrative severeinfiltration and scales and lichenification. erythema, scalesinfiltration. lichenification. crusts. and numerous excoriations.Effects of Since recurrence Out of control Sufficient RecurrenceSufficient steroid soon observed by with steroid effects are notoccurred soon effects are not external discontinuation external obtainedwith after obtained with drug of steroid therapy. steroid externaldiscontinuation steroid external external drugs for of steroid drugs fortherapy, non- erythema and external lichenified steroid externalinfiltrative therapy. lesions and preparations erythema on theinfiltrative were applied, face. erythema. leading to furtherexacerbation. Evaluation Severe Most severe Severe Severe Severe ofsymptoms

TABLE 12 Therapeutic effects of CNP ointment preparation Subject Subject21 Subject 23 (FIG. 6) Subject 22 (FIG. 7) Subject 24 Subject 25 Case —— — — — Sex Female Male Female Female Male Age 21 years old 21 years old28 years old 33 years old 39 years old Dosage form CNP ointment CNPointment CNP ointment CNP ointment CNP ointment preparation preparationpreparation preparation preparation Dosage 30 μg/g 50 μg/g 50 μg/g 50μg/g 50 μg/g Number of Twice a day Twice a day Twice a day Twice a dayTwice a day administration Days of 2 days 2 days 3 days 2 days 3 daysadministration Applied region Face and neck Face and neck Face and neckFace and neck Face and neck Severity level Before: severe Before: severeBefore: severe Before: severe Before: severe of rash by After: mildAfter: mild After: mild After: mild After: mild DermatologicalAssociation Guideline Severity level Erythema: 3 Erythema: 3 Erythema: 3Erythema: 3 Erythema: 2 evaluation of Edema/papulation: 2Edema/papulation: 1 Edema/papulation: 2 Edema/papulation: 2Edema/papulation: 1 the rash Oozing/crusting: 3 Oozing/crusting: 2Oozing/crusting: 3 Oozing/crusting: 2 Oozing/crusting: 1 region byExcoriation: 2 Excoriation: 1 Excoriation: 2 Excoriation: 1 Excoriation:1 SCORAD (before Lichenification: 2 Lichenification: 2 Lichenification:2 Lichenification: 2 Lichenification: 2 application) Dryness: 3 Dryness:3 Dryness: 3 Dryness: 2 Dryness: 3 Total: 15/18 Total: 12/18 Total:15/18 Total: 12/18 Total: 10/18 Severity level Erythema: 1 Erythema: 1Erythema: 1 Erythema: 1 Erythema: 1 evaluation of Edema/papulation: 1Edema/papulation: 0 Edema/papulation: 1 Edema/papulation: 1Edema/papulation: 0 the rash Oozing/crusting: 1 Oozing/crusting: 1Oozing/crusting: 1 Oozing/crusting: 0 Oozing/crusting: 0 region byExcoriation: 1 Excoriation: 1 Excoriation: 0 Excoriation: 0 Excoriation:0 SCORAD (after Lichenification: 1 Lichenification: 1 Lichenification: 1Lichenification: 1 Lichenification: 1 application) Dryness: 1 Dryness: 1Dryness: 1 Dryness: 1 Dryness: 1 Total: 6/18 Total: 5/18 Total: 5/18Total: 4/18 Total: 3/18 Detailed After 2 days of After 2 days of After 3days of After 2 days of After 3 days of description of application ofapplication to application to application to application to improvement30-μg/g CNP the face and neck the face and neck the face and neck theface and neck status of ointment twice a day, twice a day, twice a day,twice a day, symptoms preparation to erythema, erythema, erythema,erythema, the face twice a infiltration, and excoriations, infiltration,and infiltration, and day, erythema, itching were infiltration, anditching were itching were infiltration, markedly itching were markedlymarkedly scales and improved. markedly improved. improved. itching wereimproved. markedly improved. Itching Before: 10 Before: 10 Before: 10Before: 10 Before: 10 sensation After: 0 After: 1 After: 0 After: 0After: 0 Non-recurrence 5 days 7 days 7 days 10 days 2 weeks period

TABLE 13 Diagnosis prior to application of CNP ointment preparation.Subject Subject 27 Subject 28 Subject 29 Subject 30 Subject 26 (FIG. 10)(FIG. 11) (FIG. 8) (FIG. 9) Case — — — — — Sex Female Female Female MaleMale Age 32 years old 3 years old 22 years old 44 years old 36 years oldOnset and Erythema began Developed at 2 Developed in Starting 10Developed in course of to appear on the months of age, infancy, havingyears ago, infancy, having disease face at the age having erythemarecurrent eczema chronic recurrent eczema of 20; erythema over the wholewith itching; recurrent with itching; and papules body. In due tosteroid dermatitis with due to steroid appeared on the particular, theexternal itching external back and four face is application repeatedlyapplication limbs, which sometimes full every day, skin appeared due toevery day worsened by of excoriations flush, scales, the stress starting10 sweating; they due to and infiltrative caused by years ago, skin haverecurred scratching all erythema become professional flush, scalesrepeatedly. the time. observed over responsibility. and infiltrated thewhole body. erythema She is appeared over complicated with the wholebody. intractable alopecia. Family Elder brother; Father; Atopic Mother;Allergic Child; Atopic history Atopic dermatitis rhinitis dermatitisdermatitis Elder brother: Atopic dermatitis Past Allergic AllergicAllergic Allergic history rhinitis, rhinitis, rhinitis rhinitisConjunctivitis Scratch House dust: 2+ House dust: 1+ House dust: 2+House dust: 1+ House dust: 2+ test Mite: 2+ Mite: 2+ Mite: 3+ Mite: 3+Mite: 1+ Cedar: 2+ Egg white: 2+ Cedar: 3+ Cedar: 1+ Cedar: 2+ Orchardgrass: 3+ Ragweed: 1+ Orchard grass: 1+ Ragweed: 2+ DiagnosticInfiltrative Infiltrative Infiltrative Infiltrative Infiltrativefindings erythema and erythema, erythema, erythema, erythema, swellingof the lichenified lichenified lichenified lichenified face and necklesions, lesions, lesions, lesions, are observed, erythema, severeerythema, severe erythema, erythema, severe and erythema, scales,adhesion scales, and adhesion of scales, adhesion scales, adhesion ofcrusts, and adhesion of crusts and of crusts and of crusts, and numerouscrusts are numerous numerous numerous excoriations are observed overexcoriations are excoriations are excoriations are observed over thewhole body. observed on the observed over observed on the the wholebody. She is in a body trunk. the whole body. four limbs and She is in astate of He is in a state body trunk. state of erythroderma oferythroderma erythroderma posteczematosa. posteczematosa.posteczematosa. Symptoms of Rash on the face Rash on the face Rash onthe face Rash on the back Rash on the face application and neck isconsists of and neck is forms a plaque and upper limbs regions mainlyinfiltration/ mainly accompanied by is mainly characterized by severescales, characterized by erythema with characterized by erythema withadhesion of infiltrative infiltration/ erythema with swelling. crusts,erythema with lichenification, severe swelling erosions, and swelling.scales, and and numerous adhesion of infiltration. excoriations. crusts.Effects of Symptoms recur After Not improved Recurrence Not improvedsteroid soon after discontinuation even by “very appears soon even bysteroid external discontinuation of steroid strong” steroid with “veryexternal drug of steroid external external strong” steroid therapy.external application, therapy. external therapy. skin flush and therapy.desquamation appear over the whole body, and she becomes a state oferythroderma. Evaluation Severe Most severe Most severe Severe Mostsevere of symptoms

TABLE 14 Therapeutic effects of CNP ointment preparation. SubjectSubject 27 Subject 28 Subject 29 Subject 30 Subject 26 (FIG. 10) (FIG.11) (FIG. 8) (FIG. 9) Case — — — — — Sex Female Female Female Male MaleAge 32 years old 3 years old 22 years old 44 years old 36 years oldDosage form CNP ointment CNP ointment CNP ointment CNP ointment CNPointment preparation preparation preparation preparation preparationDosage 50 μg/g 50 μg/g 50 μg/g 50 μg/g 50 μg/g Number of Twice a dayTwice a day Twice a day Twice a day Twice a day administration Days of 2days 3 days 3 days 3 days 2 days administration Applied region Face andneck Face Face, neck, head Neck and back Face and upper limbs Severitylevel Before: severe Before: severe Before: severe Before: severeBefore: severe of rash by After: mild After: mild After: minor After:mild After: mild Dermatological Association Guideline Severity levelErythema: 3 Erythema: 3 Erythema: 3 Erythema: 3 Erythema: 3 evaluationof Edema/papulation: 2 Edema/papulation: 2 Edema/papulation: 3Edema/papulation: 2 Edema/papulation: 3 the rash Oozing/crusting: 1Oozing/crusting: 3 Oozing/crusting: 3 Oozing/crusting: 3Oozing/crusting: 3 region by Excoriation: 1 Excoriation: 3 Excoriation:2 Excoriation: 2 Excoriation: 3 SCORAD (before Lichenification: 1Lichenification: 2 Lichenification: 2 Lichenification: 3Lichenification: 3 application) Dryness: 2 Dryness: 3 Dryness: 3Dryness: 3 Dryness: 3 Total: 10/18 Total: 16/18 Total: 16/18 Total:16/18 Total: 18/18 Severity level Erythema: 1 Erythema: 1 Erythema: 0Erythema: 1 Erythema: 1 evaluation of Edema/papulation: 1Edema/papulation: 1 Edema/papulation: 0 Edema/papulation: 1Edema/papulation: 0 the rash Oozing/crusting: 1 Oozing/crusting: 1Oozing/crusting: 1 Oozing/crusting: 1 Oozing/crusting: 1 region byExcoriation: 0 Excoriation: 1 Excoriation: 0 Excoriation: 1 Excoriation:1 SCORAD (after Lichenification: 0 Lichenification: 1 Lichenification: 0Lichenification: 1 Lichenification: 1 application) Dryness: 1 Dryness: 1Dryness: 1 Dryness: 1 Dryness: 1 Total: 4/18 Total: 6/18 Total: 2/18Total: 6/18 Total: 5/18 Detailed After 2 days of After 3 days of After 7days of After 3 days of After 2 days of description of application tothe application to the application to the application to the applicationto the improvement face and neck face twice a day, face, neck and neckand back face and upper status of twice a day, erythema, head twice aday, twice a day, limbs twice a day, symptoms erythema, swellinginfiltration, erythema, lichenification, erythema, and itching werescales, and infiltration, and erythema, excoriations, improved. itchingwere itching were infiltration, infiltration and markedly improved.improved, and scales and itching itching were remarkable hair weremarkedly markedly improved. growth was improved. observed in thehair-loss regions. Itching Before: 10 Before: 10 Before: 10 Before: 10Before: 10 sensation After: 2 After: 1 After: 1 After: 1 After: 1Non-recurrence 5 days 2 weeks 2 weeks 2 weeks or more 5 days period

TABLE 15 Diagnosis prior to application of CNP ointment preparation.Subject Subject 31 Subject 32 Subject 33 Subject 34 Subject 35 Case — —— — — Sex Female Female Female Male Male Age 38 years old 24 years old56 years old 36 years old 25 years old Onset and Developed in ChronicSkin flush with Developed in Diagnosed to course of infancy; recurrent aburning infancy, having have atopic disease worsening of dermatitis withsensation recurrent eczema dermatitis at 2 symptoms was itching occurrederythema, and with itching; months of age; triggered by repeatedly sincescales appeared since 4 years since then he delivery 7 yearskindergarten; on the face ago, steroid has continued to ago, and due toexternal since several external drugs visit erythema with application ofyears ago, and have been dermatology itching appeared strong steroidthey recurred applied every department for mainly on the ointments, sherepeatedly. day, resulting treatment, but face, upper has erythrodermain skin flush, symptoms limbs and body posteczematosa; scales andworsened trunk; despite she has had infiltrative starting 2 yearsapplication of cataract surgery erythema over ago; he has steroidexternal and retinal the whole body. erythroderma drugs, detachment 6posteczematosa recurrence times, and she presumably due persisted. useseye-drops to long-term to decrease eye steroid external pressure.therapy. Family Child; Atopic Father, mother; Elder brother; Mother;Atopic history dermatitis Allergic Atopic dermatitis rhinitisdermatitis, Younger sister; Bronchial asthma Atopic dermatitis PastAllergic Allergic Allergic Allergic Child asthma, history rhinitis,rhinitis rhinitis rhinitis Allergic Allergic rhinitis, conjunctivitisAllergic conjunctivitis Scratch House dust: 2+ House dust: 3+ Housedust: 1+ House dust: 2+ House dust: 2+ test Mite: 2+ Mite: 3+ Mite: 2+Mite: 3+ Mite: 3+ Cedar: 1+ Cedar: 3+ Orchard grass: 1+ Cedar: 1+Orchard grass: 2+ Orchard grass: 2+ Orchard grass: 1+ Orchard grass: 3+Ragweed: 2+ Ragweed: 2+ Ragweed: 1+ Diagnostic Infiltrative InfiltrativeInfiltrative Infiltrative Strong itching findings erythema witherythema, erythema, erythema, with sleep itching lichenified lichenifiedlichenified disturbance, associated with lesions, lesions, lesions,infiltrative sleep erythema, severe erythema, severe erythema, severeerythema, disturbance, and scales, adhesion scales, adhesion scales,adhesion lichenified erythema are of crusts, and of crusts, and ofcrusts, and lesions and observed on numerous numerous numerous erythemaare almost whole excoriations are excoriations are excoriations areobserved over body. observed over observed on the observed over thewhole body; the whole body. four limbs and the whole body. and severeShe has face. He has scales, erythroderma erythroderma infiltrativeposteczematosa. posteczematosa. erythema, adhesion of crusts, andnumerous excoriations are observed on the face. Symptoms of Rash on theface Rash on the face Rash on the face Rash on the face Rash on the faceapplication is mainly includes is mainly is mainly is mainly regionscharacterized by erythema with characterized by characterized bycharacterized by erythema with severe swelling/ erythema, erythema withsevere severe edema/ erosions, lichenification, infiltration,infiltration and infiltration/or crusts, and erosions, erythema, edema.lichenification, excoriations. scales, and exudate, crusts, as well asnumerous and numerous severe scales, excoriations. excoriations.adhesion of crusts, vesicles, erosions, and numerous excoriations.Effects of Steroid external Skin flush and Infiltrative Not improved Hehas steroid drug is not desquamation erythema on the even by “veryerythroderma external sufficiently appear over the face is strong”steroid posteczematosa drug effective whole body upon temporarilyexternal presumably due against discontinuation reduced by therapy. tosteroid erythema, of steroid steroid external external infiltrativeexternal therapy, but therapy. erythema and application, symptoms soonscales on the leading to the recur with a face, upper state of worseninglimbs and body erythroderma. tendency. trunk, which show an intractablenature. Evaluation Severe Most severe Moderate Most severe Most severeof symptoms

TABLE 16 Therapeutic effects of CNP ointment preparation Subject Subject31 Subject 32 Subject 33 Subject 34 Subject 35 Case — — — — — Sex FemaleFemale Female Male Male Age 38 years old 24 years old 56 years old 36years old 25 years old Dosage form CNP ointment CNP ointment CNPointment CNP ointment CNP ointment preparation preparation preparationpreparation preparation Dosage 100 μg/g 100 μg/g 100 μg/g 100 μg/g 100μg/g Number of Twice a day Twice a day Twice a day Twice a day Twice aday administration Days of 3 days 3 days 3 days 3 days 3 daysadministration Applied region Face Face Face Face and back Face Severitylevel Before: moderate Before: severe Before: severe Before: severeBefore: severe of rash by After: minor After: moderate After: minorAfter: mild After: mild Dermatological Association Guideline Severitylevel Erythema: 2 Erythema: 3 Erythema: 3 Erythema: 2 Erythema: 2evaluation of Edema/papulation: 2 Edema/papulation: 2 Edema/papulation:2 Edema/papulation: 1 Edema/papulation: 2 the rash Oozing/crusting: 1Oozing/crusting: 3 Oozing/crusting: 3 Oozing/crusting: 2Oozing/crusting: 3 region by Excoriation: 1 Excoriation: 3 Excoriation:1 Excoriation: 2 Excoriation: 3 SCORAD (before Lichenification: 1Lichenification: 3 Lichenification: 1 Lichenification: 3Lichenification: 2 application) Dryness: 1 Dryness: 3 Dryness: 1Dryness: 3 Dryness: 2 Total: 8/18 Total: 17/18 Total: 11/18 Total: 13/18Total: 14/18 Severity level Erythema: 0 Erythema: 1 Erythema: 0Erythema: 1 Erythema: 1 evaluation of Edema/papulation: 0Edema/papulation: 1 Edema/papulation: 0 Edema/papulation: 0Edema/papulation: 1 the rash Oozing/crusting: 0 Oozing/crusting: 1Oozing/crusting: 0 Oozing/crusting: 1 Oozing/crusting: 1 region byExcoriation: 1 Excoriation: 1 Excoriation: 0 Excoriation: 0 Excoriation:1 SCORAD (after Lichenification: 0 Lichenification: 2 Lichenification: 0Lichenification: 1 Lichenification: 1 application) Dryness: 0 Dryness: 2Dryness: 1 Dryness: 1 Dryness: 1 Total: 1/18 Total: 8/18 Total: 1/18Total: 4/18 Total: 6/18 Detailed After 3 days of After 3 days of After 3days of After 3 days of After 3 days of description of application toapplication to application to application to application to improvementthe face twice a the face twice a the face twice a the face and neck theface twice a status of day, erythema and day, erythema, day, erythema,twice a day, day, erythema, symptoms infiltration were infiltration,infiltration, erythema, infiltration, markedly improved scales, andcrusts, itching, infiltration, scales, and to the best itching were andburning scales, and itching were condition since markedly sensation wereitching were markedly 20 years ago. improved. markedly markedlyimproved. improved. improved. Itching Before: 10 Before: 10 Before: 10Before: 10 Before: 10 sensation After: 2 After: 1 After: 0 After: 1After: 1 Non-recurrence 10 days 7 days 2 weeks 2 weeks or more 5 daysperiodSummary of Therapeutic Effects of CNP Gel Preparation, CNPAqueous-Solution Preparation, CNP Gel-Base Preparation, and CNP OintmentPreparation:

The above case studies clarified the following.

By administering skin external preparations comprising CNP, erythemawith severe swelling/edema/infiltration, erythema with lichenification,papules, and scales were markedly improved, or they were improved to themild symptoms that are characterized mainly by dryness, mild erythema,scales, etc., or to the minor rash characterized mainly by dryness withless inflammation. In particular, by administering skin externalpreparations comprising CNP, skin flush, infiltration, scaleslichenification and burning sensation of on the face of adult patients,which are difficult to cure and can disrupt the patients' social lives,could be dramatically improved to a condition without any irritationsymptoms. Moreover, remarkable improvement was observed in other regionssuch as the upper limbs and back, and similar effects were observed forinfants as well, and were not limited to adults. Any of these skinexternal preparations comprising CNP such as CNP gel preparation, CNPaqueous-solution preparation, CNP gel-base preparation and CNP ointmentpreparation demonstrated almost identical therapeutic effects.

Manifestation of these effects of the skin external preparationscomprising CNP is as follows: approximately 10 min after theapplication, a subjective burning sensation subsides, and on and afterapproximately 30 min after the application, improvements in erythema andinfiltration were objectively observed. Furthermore, continuation of theapplication for 3 or 4 days apparently reduced erythema andinfiltration, resulting in near-normal skin with fine texture. Thiscould be a great relief for patients with severe atopic dermatitis.

In addition, by administering skin external preparations comprising CNP,beneficial changes in vasodilation and inflammatory erythema wasobserved by the findings in the upper dermis of the skin by dermoscopy.Furthermore, by administering skin external preparations comprising CNP,it was demonstrated that the texture of the skin surface becomes finer,scales decrease, and the skin become soft to the touch. These findingswere also confirmed by the findings in the upper dermis of the skin bydermoscopy. The fact that “the texture of the skin surface becomesfiner, scales decrease, and the skin become soft to the touch” isimportant in compensating for skin dryness and deterioration of barrierfunctions, as well as preventing recurrence of inflammation. Sucheffects were also observed in psoriasis; disappearance of scales andreduction of infiltration were observed. Neither local irritationsymptoms nor systemic side effects were observed at all by the CNPapplication. Likewise in rosacea, improvements in capillary dilation anderythema were achieved by the CNP application.

These therapeutic effects of good skin conditions were maintained forbetween 5 days to approximately 2 weeks after discontinuation of theapplication. Thereafter, even when erythema relapsed, it did not worsenas was observed before the application, and symptoms were merely mild,and this demonstrates that stable conditions could be maintained. Thisdeserves a special note since these effects could not have been obtainedby conventional therapy, i.e., steroid external application. Inaddition, even when symptoms relapsed, it was evident thatre-application to the rash could lead to mild or minor rash by a smallernumber of applications than the initial application.

The primary goal of treatment of atopic dermatitis is to achieve thefollowing conditions in patients.

(1) No symptoms; if any, minor symptoms without any problems caused indaily life, with a requirement of a low degree of drug therapy.

(2) While minor or mild symptoms persist, there is a small possibilityof acute worsening; even when the symptoms worsen, they will not persistfor a long time.

The above case studies clearly demonstrated that the skin externalpreparations comprising CNP of the present invention are able to achievethese conditions in patients.

Next, skin external preparations comprising BNP were examined.

EXAMPLE 13

1. Production of BNP Gel-Base Preparation:

Preparation of the BNP gel-base preparations was performed as follows.

0.1 g of methyl parahydroxybenzoate (product name: Mekkins M, Ueno FineChemicals Industry), 0.2 g of phenoxyethanol, and 3.0 g of1,2-pentanediol were measured in the same container, dissolved at 60-70°C., and introduced into a mixing kettle. 6.0 g of concentrated glycerinwas introduced, and a mixture of 0.44 g of carboxy vinyl polymer(product name: Carbopol 940, Lubrisol Advanced Materials, Inc.) and 0.08g of xanthan gum (product name: Keltrol T, CP Kelco) was added to thissolution and stirred thoroughly with a paddle at 15 rpm for dispersion.Then, while stirring with a paddle at 15 rpm, 83.95 g of purified waterwas gradually introduced, and the mixture was dissolved by stirring at akettle temperature of 70-80° C. using a paddle at 20 rpm and a disperserat 1500-2000 rpm. After stopping the disperser, dissolution wasconfirmed and cooling was immediately started; when the kettletemperature approached around 40° C., 6.0 g of Lubrajel NP from ISPJapan, Ltd. (2.7 g of glycerin, 0.06 g of carboxy vinyl polymer, 0.018 gof sodium polyacrylate, 3.222 g of water) was added and mixedhomogeneously with a paddle at 20 rpm, then 0.230 g of potassiumhydroxide was added for neutralization, and when the kettle temperaturereached 25° C., the rotation of the paddle was terminated to obtain agel-base.

Then, 3 mg of human BNP-32 (Peptide Institute, Inc.) as a principalagent was dissolved in 3 ml of saline to obtain the BNP solution with aconcentration of 1000 μg/ml, and 1 ml of this BNP solution washomogeneously stirred and mixed in 19 g of the gel-base obtained asabove, to produce the gel-base preparation with a concentration of 50μg/g.

Similarly, 600 μl of the above BNP solution with a concentration of 1000μg/ml was diluted with 400 μl of saline to adjust the concentration at600 μg/ml, then 1 ml of the resulting solution was homogeneously mixedin 19 g of the gel-base obtained as above and stirred, to produce thegel-base preparation with a concentration of 30 μg/g.

EXAMPLE 14

2. Production of BNP Aqueous-Solution Preparation:

3 mg of human BNP-32 (Peptide Institute, Inc.) as a principal agent wasdissolved in 3 ml of saline to obtain a BNP solution, and 1 ml of thissolution was diluted with 19 ml of saline to produce theaqueous-solution preparation with a BNP concentration of 50 μg/ml.

EXAMPLE 15

3. Diagnosis of Subjects:

The patients′ diagnosis, evaluation of symptoms, selection of externaltherapy, test methods and observation of the skin were performedsimilarly to the methods of Example 1.

In addition, similarly to the methods of Example 1, prior toadministration of the BNP gel-base preparation or BNP aqueous-solutionpreparation of the present invention, history taking from the subjects,scratch tests for allergens and diagnosis were conducted. Table 17(subjects 36-40) and Table 19 (subjects 41-45) show the results of thesubjects′ history taking and diagnosis, i.e., sex, age, onset and courseof disease, family history, past history, scratch test results,diagnostic findings, and symptom evaluation based on “Guideline 2005” ofthe subject in each case.

EXAMPLE 16

4. Therapeutic Effects on Subjects:

Therapeutic effects of the BNP gel-base preparation or BNPaqueous-solution preparation of the present invention are shown in Table18 (subjects 36-40) and Table 20 (subjects 41-45). In Tables 18 and 20,“itching sensation” represents a comparison of the itching sensationevaluated using the visual analogue scale method before and aftertreatment. Similarly, “non-recurrence period” refers to the period afterdiscontinuation of the treatment by the preparation of the presentinvention subsequent to improvement of symptoms, for which relapse ofthe symptoms did not occur. In order to evaluate objectively,photographs before and after the application of BNP preparations weretaken for all cases. Of these, photographs of some of the cases areshown in the figures.

TABLE 17 Diagnosis prior to application of BNP gel-base preparation orBNP aqueous-solution preparation. Subject Subject 40 Subject 36 Subject37 Subject 38 Subject 39 (FIG. 14) Case Case 7 Case 6 Case 2 Case 3 Case5 Sex Female Female Male Male Female Age 32 years old 22 years old 31years old 21 years old 36 years old Onset and She had severe Developedsoon Developed atopic Developed in Developed in course of bronchialasthma after birth, dermatitis in infancy; infancy, disease until about20 continuously infancy; symptoms continuously years of age; receivinginfiltrative worsened after using since then, she steroid externalerythema entering commercially- has been therapy; appeared university 3available suffering mainly symptoms particularly on years ago andsteroid from erythema on worsened in the face eczema is ointments; butthe face. junior-high recently, appearing even the symptoms do school.Symptoms showing on the face. not subside at relapse soon intractableall, and she can after nature. hardly sleep discontinuation because ofdue to steroid itching. rebound, and worsen further. Family Elderbrother; Uncle; Allergic Mother; Allergic Father; Father; Atopic historyAtopic rhinitis rhinitis Bronchial asthma dermatitis, dermatitisAllergic rhinitis Past Allergic Allergic Allergic Bronchial asthmaAllergic history rhinitis, rhinitis rhinitis rhinitis ConjunctivitisScratch House dust: 3+ House dust: 2+ House dust: 1+ House dust: 2+House dust: 2+ test Mite: 3+ Mite: 2+ Mite: 3+ Mite: 3+ Mite: 3+ Cedar:2+ Cedar: 1+ Cedar: 3+ Cedar: 2+ Cedar: 2+ Orchard grass: 2+ Orchardgrass: 2+ Orchard grass: 3+ Ragweed: 1+ Ragweed: 1+ Ragweed: 2+Diagnostic Infiltrative Edema, Infiltrative Infiltrative Infiltrativefindings erythema, edema, infiltrative erythema, erythema, erythema, anderythema are erythema, erythema and erythema, many, erythema, edema,disseminated on erythema, scales on the adhesion of scales, adhesion theface and scales, and face, neck and crusts, and of crusts, and neck;papules numerous body trunk. excoriations are numerous and erythema areexcoriations are observed on the excoriations are disseminated onobserved over face, four observed on the the four limbs. the whole body.limbs, and back. face, neck, four She has limbs and body erythrodermatrunk. posteczematosa. Symptoms of Rash on the face Rash on the faceRash on the face Rash on the face Rash on the face application and neckis and neck is and neck is is mainly and neck is regions mainly mainlymainly characterized by mainly characterized by characterized bycharacterized by infiltrative characterized by severe severe edema,severe erythema, many erythema with infiltration, infiltration,infiltration, papules, and edema, erosions, erythema and erythema,erythema and excoriations. scales and swelling. erosions, scales scales.numerous and numerous excoriations. excoriations. Effects of Symptomsrelapse Symptoms cannot Symptoms relapse Symptoms relapse Symptoms arenot steroid soon after be improved by soon after soon after reduced byexternal discontinuation steroid external discontinuationdiscontinuation steroid external drug of steroid therapy. of steroid ofsteroid therapy. external external external therapy. therapy. therapy.Evaluation Severe Most severe Severe Severe Most severe of symptoms

TABLE 18 Therapeutic effects of BNP gel-base preparation or BNPaqueous-solution preparation. Subject Subject 40 Subject 36 Subject 37Subject 38 Subject 39 (FIG. 14) Case Case 7 Case 6 Case 2 Case 3 Case 5Sex Female Female Male Male Female Age 32 years old 22 years old 31years old 21 years old 36 years old Dosage form BNP gel-base BNPgel-base BNP gel-base BNP gel-base BNP gel-base preparation preparationpreparation preparation preparation Dosage 30 μg/g 30 μg/g 50 μg/g 50μg/g 50 μg/g Number of Twice a day Twice a day Twice a day Twice a dayTwice a day administration Days of 3 days 5 days 3 days 3 days 2 daysadministration Applied region Face Face and neck Face Face Face and neckSeverity level Before: severe Before: severe Before: severe Before:severe Before: severe of rash by After: minor After: mild After: minorAfter: mild After: mild Dermatological Association Guideline Severitylevel Erythema: 3 Erythema: 3 Erythema: 3 Erythema: 3 Erythema: 3evaluation of Edema/papulation: 3 Edema/papulation: 3 Edema/papulation:2 Edema/papulation: 2 Edema/papulation: 3 the rash Oozing/crusting: 1Oozing/crusting: 2 Oozing/crusting: 2 Oozing/crusting: 2Oozing/crusting: 1 region by Excoriation: 1 Excoriation: 2 Excoriation:2 Excoriation: 2 Excoriation: 2 SCORAD (before Lichenification: 1Lichenification: 2 Lichenification: 1 Lichenification: 1Lichenification: 2 application) Dryness: 2 Dryness: 3 Dryness: 2Dryness: 2 Dryness: 3 Total: 11/18 Total: 15/18 Total: 12/18 Total:12/18 Total: 14/18 Severity level Erythema: 1 Erythema: 1 Erythema: 1Erythema: 1 Erythema: 1 evaluation of Edema/papulation: 0Edema/papulation: 1 Edema/papulation: 0 Edema/papulation: 1Edema/papulation: 1 the rash Oozing/crusting: 0 Oozing/crusting: 1Oozing/crusting: 0 Oozing/crusting: 1 Oozing/crusting: 0 region byExcoriation: 0 Excoriation: 0 Excoriation: 0 Excoriation: 0 Excoriation:0 SCORAD (after Lichenification: 0 Lichenification: 0 Lichenification: 0Lichenification: 0 Lichenification: 0 application) Dryness: 0 Dryness: 1Dryness: 1 Dryness: 1 Dryness: 1 Total: 1/18 Total: 4/18 Total: 2/18Total: 4/18 Total: 3/18 Detailed After 2 to 3 days After 5 days of After3 days of After 3 days of By application of description of ofapplication of application of 30 μg/g application of 50 μg/g applicationof 50 μg/g 50 μg/g BNP gel- improvement 30 μg/g BNP gel- BNP gel-baseBNP gel-base BNP gel-base base preparation status of base preparation,preparation to preparation to preparation to twice a day, symptomserythema, edema, the face and neck the face twice a the face twice aerosions were dryness and twice a day, day, erythema, day, erythema,improved in 1 itching were edema, erythema, infiltration, infiltration,and day, and 2 days markedly excoriations, burning sensation itchingwere later, erythema, improved. infiltration, and itching were markedlyinfiltration, scales, burning markedly improved. excoriations andsensation and improved. itching were itching were Symptoms are markedlymarkedly mainly improved. improved. characterized by dryness with lessinflammatory symptoms. Itching Before: 10 Before: 10 Before: 10 Before:10 Before: 10 After: 0 After: 2 After: 0 After: 3 After: 1Non-recurrence 10 days 5 days 2 weeks 2 weeks 7 days period

TABLE 19 Diagnosis prior to application of BNP gel-base preparation orBNP aqueous-solution preparation. Subject Subject 41 Subject 42 Subject43 (FIG. 12) (FIG. 13) (FIG. 15) Subject 44 Subject 45 Case Case 1 Case4 Case 8 Case 9 Case 10 Sex Female Male Male Male Female Age 11 yearsold 23 years old 21 years old 28 years old 48 years old Onset andDeveloped in Developed at Symptoms Developed when Developed at 3 courseof infancy; having around 3 years worsened since he was in months ofage; disease recurrent eczema of age; symptoms last autumn, andelementary symptoms worsen that worsens as worsened after he has eczemaschool. In as seasons seasons change; transferring over the whole recentyears change. Recently it developed jobs last year. body includingintractable the rash into In particular the face, and he recurrentworsened due to erythroderma erythema on the claims that he erythemabecame leisureless recently. face shows can hardly sleep obvious mainlylife, and she is intractable because of on the face. in a state ofnature. itching. erythroderma. Family Mother; Atopic Mother; AllergicMother; Elder brother; history dermatitis rhinitis Bronchial asthmaAtopic dermatitis Past Allergic Bronchial asthma Allergic Allergichistory rhinitis rhinitis rhinitis Scratch House dust: 2+ House dust: 2+House dust: 3+ House dust: 1+ House dust: 3+ test Mite: 3+ Mite: 3+Mite: 3+ Mite: 1+ Mite: 3+ Cedar: 2+ Cedar: 2+ Cedar: 2+ Cedar: 1+Cedar: 2+ Orchard grass: 2+ Orchard grass: 2+ Orchard grass: 2+ Orchardgrass: 2+ Orchard grass: 3+ Ragweed: 1+ Ragweed: 1+ Ragweed: 3+Diagnostic Edema, Infiltrative Infiltrative Infiltrative Skin flush withfindings infiltrative erythema, erythema with erythema, chills, edema,erythema, erythema, edema, lichenification, erythema, edema, andinfiltrative erythema, severe scales, and erythema, many scales, anderythema are scales, and adhesion of papules and numerous observed onthe numerous crusts are excoriations are excoriations are almost wholeexcoriations are observed on the observed on the observed on the body.observed over four limbs, body body; and four limbs, body the wholebody. trunk, neck and infiltrative trunk, neck and She has face.erythema, face. erythroderma erythema and posteczematosa. many papulesare observed on the face. Symptoms of Rash on the neck Rash on the Rashon the face Rash on the face Rash on the application is mainly upperlimbs is mainly consists of face, neck, and regions characterized byconsists of characterized by infiltration, four limbs severe edema,infiltrative infiltrative erythema, severe consist of infiltration,erythema, erythema, many scales and severe swelling, erythema, erythema,papules, scales crusts. skin flush and erosions, papules, scales andedema. scales, and and crusts. excoriations. numerous excoriations.Effects of Symptoms cannot Symptoms cannot Symptoms relapse Symptomssoon She is in a steroid be improved by be controlled by soon afterrelapse with state of external steroid external steroid externaldiscontinuation steroid external erythroderma due drug therapy.application. of steroid therapy. to side effects external of long-termand therapy. excessive steroid external application. Evaluation Mostsevere Most severe Most severe Severe Most severe of symptoms

TABLE 20 Therapeutic effects of BNP gel-base preparation or BNPaqueous-solution preparation. Subject Subject 41 Subject 42 Subject 43(FIG. 12) (FIG. 13) (FIG. 15) Subject 44 Subject 45 Case Case 1 Case 4Case 8 Case 9 Case 10 Sex Female Male Male Male Female Age 11 years old23 years old 21 years old 28 years old 48 years old Dosage form BNPgel-base BNP gel-base BNP aqueous- BNP aqueous- BNP aqueous- preparationpreparation solution solution solution preparation preparationpreparation Dosage 50 μg/g 50 μg/g 50 μg/ml 50 μg/ml 50 μg/ml Number ofTwice a day Twice a day Twice a day Twice a day Twice a dayadministration Days of 5 days 5 days 5 days 1 day 3 days administrationApplied region Neck Upper limbs Face Face and neck Face and neckSeverity level Before: severe Before: severe Before: severe Before:severe Before: severe of rash by After: mild After: mild After: mildAfter: minor After: moderate Dermatological Association GuidelineSeverity level Erythema: 3 Erythema: 2 Erythema: 3 Erythema: 3 Erythema:3 evaluation of Edema/papulation: 3 Edema/papulation: 2Edema/papulation: 2 Edema/papulation: 2 Edema/papulation: 3 the rashOozing/crusting: 3 Oozing/crusting: 2 Oozing/crusting: 2Oozing/crusting: 3 Oozing/crusting: 3 region by Excoriation: 3Excoriation: 2 Excoriation: 1 Excoriation: 2 Excoriation: 2 SCORAD(before Lichenification: 3 Lichenification: 2 Lichenification: 2Lichenification: 2 Lichenification: 3 application) Dryness: 2 Dryness: 3Dryness: 2 Dryness: 2 Dryness: 3 Total: 17/18 Total: 13/18 Total: 12/18Total: 14/18 Total: 17/18 Severity level Erythema: 1 Erythema: 1Erythema: 1 Erythema: 1 Erythema: 2 evaluation of Edema/papulation: 1Edema/papulation: 0 Edema/papulation: 0 Edema/papulation: 0Edema/papulation: 1 the rash Oozing/crusting: 1 Oozing/crusting: 1Oozing/crusting: 1 Oozing/crusting: 0 Oozing/crusting: 2 region byExcoriation: 1 Excoriation: 0 Excoriation: 0 Excoriation: 0 Excoriation:1 SCORAD (after Lichenification: 1 Lichenification: 0 Lichenification: 0Lichenification: 0 Lichenification: 1 application) Dryness: 1 Dryness: 1Dryness: 1 Dryness: 1 Dryness: 2 Total: 6/18 Total: 3/18 Total: 3/18Total: 2/18 Total: 9/18 Detailed After 5 days of After 3 days of After 2days of By the By the description of application of 50 μg/g applicationof 50 μg/g application of 50 μg/ml application of 50 μg/ml applicationof 50 μg/ml improvement BNP gel-base BNP gel-base BNP saline- BNPsaline- BNP saline- status of preparation to preparation twice solutionsolution solution symptoms the neck twice a a day, erythema, preparationtwice preparation twice preparation twice day, edema, infiltration, anda day, erythema, a day, on the a day, swelling, erythema, itching wereinfiltration, next day, erythema, itching excoriations, markedlyimproved scales and erythema, itching sensation and infiltration,although the skin itching were and dryness were tight-stretched scales,burning was slightly dry. markedly markedly sensation were sensation andimproved. improved. fairly improved itching were in 1 day, and markedlyinfiltrative improved. erythema, Symptoms are swelling, mainlyexcoriations and characterized by itching were dryness, mild markedlyimproved erythema, and 3 days later; scales. however, scales and drynessstill remained. Itching Before: 10 Before: 10 Before: 10 Before: 10Before: 10 sensation After: 2 After: 1 After: 3 After: 0 After: 1Non-recurrence 5 days 2 weeks 10 days 7 days 5 days period

EXAMPLE 17

Details of the test examples of the BNP gel-base preparations and BNPaqueous-solution preparations shown in Tables 17-20 are described below.For reference, photographs of Case 1 (subject 41), Case 4 (subject 42),and Case 5 (subject 40) as examples of most severe patients before andafter the application are shown in FIGS. 12, 13 and 14, respectively.

BNP test example 1 (Case 1; subject 41):

The subject of Case 1 presented with edema, infiltrative erythema,erythema, severe scales, and numerous excoriations over the whole body,and was diagnosed to be in the state of erythroderma posteczematosa.Furthermore, the rash on the neck was characterized mainly by severeedema, infiltration, erythema, erosions, scales and numerousexcoriations, and the severity level based on “DermatologicalAssociation Guideline” was severe. Meanwhile, symptom evaluationaccording to “Guideline 2005” was most severe. The symptoms of thissubject did not improve by the use of steroid external preparations.

Treatment and its Outcome:

To the subject of Case 1, the BNP gel-base preparation with aconcentration of 50 μg/g obtained in Example 13 was applied to the necktwice a day; 5 days later, edema, erythema, excoriations, infiltration,scales, burning sensation and itching were markedly improved. Itchingsensation level was improved from 10 prior to the application to 2 afterthe application. FIG. 12 shows photographs before and after 5 days ofapplication; A shows the state before application, and B shows the stateafter application.

BNP test example 2 (Case 2; subject 38):

The subject of case 2 presented with infiltrative erythema, erythema andscales on the face, neck and body trunk; and the rash on the face andneck was mainly characterized by severe infiltration, erythema andscales. The rash on the face and neck was mainly characterized by severeinfiltration, erythema and scales, and the severity level based on“Dermatological Association Guideline” was severe. Meanwhile, symptomevaluation according to “Guideline 2005” was severe. Steroid externaltherapy was applied to this subject, but the subject had recurrence soonafter discontinuation of the external application.

Treatment and its Outcome:

To the subject of Case 2, the BNP gel-base preparation with aconcentration of 50 μg/g obtained in Example 13 was applied to the facetwice a day; 3 days later, erythema, infiltration, burning sensation anditching were markedly improved. Itching sensation level was improvedfrom 10 prior to the application to 0 after the application.

BNP test example 3(Case 3; subject 39):

The subject of Case 3 presented with infiltrative erythema, erythema,many papules, adhesion of crusts and excoriations on the face, fourlimbs and back, and the rash on the face was mainly characterized byinfiltrative erythema, many papules and excoriations. In addition, therash on the face and neck was mainly characterized by severeinfiltration, erythema and scales, and the severity level based on“Dermatological Association Guideline” was severe. Meanwhile, symptomevaluation based on “Guideline 2005” was severe. Steroid externaltherapy was applied to this subject; but the subject had recurrence soonafter discontinuation of the external application.

Treatment and its Outcome:

To the subject of Case 3, the BNP gel-base preparation with aconcentration of 50 μg/g obtained in Example 13 was applied to the facetwice a day; 3 days later, erythema, infiltration, and itching markedlyimproved. Itching level improved from 10 prior to the application to 0after the application for 3 days.

BNP test example 4 (Case 4; subject 42):

The subject of Case 4 presented with infiltrative erythema, erythema,edema, scales and adhesion of crusts on the four limbs, body trunk, neckand face, and the rash on the upper limbs consists of infiltrativeerythema, erythema, papules, severe scales and crusts. In addition, therash on the upper limbs consists of infiltration, erythema, edema,severe scales and crusts, and the severity level based on the“Dermatological Association Guideline” was severe. Meanwhile, symptomevaluation based on the “Guideline 2005” was most severe, and thesymptoms were not improved by steroid external preparations.

Treatment and its Outcome:

The BNP gel-base preparation with a concentration of 50 μg/g obtained inExample 13 was applied to the upper limbs of the subject of Case 4 twicea day; 3 days later, although mild erythema remained, infiltration,papules, scales, crusts and itching were markedly improved. Itchingsensation level was improved from 10 prior to the application to 1 afterthe application for 3 days. FIG. 13 shows photographs before and after 5days of application. A shows the state before application, and B showsthe state after application.

The subject of Case 5 presented with infiltrative erythema, erythema,edema, scales, adhesion of crusts and

BNP test example 5 (Case 5; Subject 40): numerous excoriations on theface, neck, four limbs and body trunk, and the rash on the face and neckwas mainly characterized by erythema with edema, erosions, scales, andnumerous excoriations. In addition, the rash on the face and neck wasmainly characterized by erythema with edema, erosions, scales, andnumerous excoriations, and the severity level based on the“Dermatological Association Guideline” was severe. Meanwhile, symptomevaluation based on the “Guideline 2005” was most severe, and thesymptoms were not improved by steroid external preparations.Treatment and its Outcome:

The BNP gel-base preparation with a concentration of 50 μg/g obtained inExample 13 was applied to the face and neck of the subject of Case 5twice a day; erosions were improved 1 day later, and erythema,infiltration, excoriations and itching were markedly improved 2 dayslater. Itching sensation level was improved from 10 prior to theapplication to 1 after the application of 2 days. FIG. 14 showsphotographs before and after 1 day of application. A shows the statebefore application, and B shows the state after application.

BNP test example 6 (Case 6; subject 37):

The subject of Case 6 presented with edema, infiltrative erythema,erythema, severe scales, and numerous excoriations over the whole body,and the subject was in a state of erythroderma posteczematosa. The rashon the face and neck was mainly characterized by severe edema,infiltration, erythema, erosions, scales and numerous excoriations, andthe severity level based on the “Dermatological Association Guideline”was severe. Meanwhile, symptom evaluation based on the “Guideline 2005”was most severe, and the symptoms of this subject were not improved bysteroid external therapy.

Treatment and its Outcome:

The BNP gel-base preparation with a concentration of 30 μg/g obtained inExample 13 was applied to the face and neck of the subject of Case 6twice a day; 5 days later, edema, erythema, excoriations, infiltration,scales, burning sensation and itching were markedly improved. Itchingsensation level was improved from 10 prior to the application to 2.

BNP test example 7 (Case 7; subject 36):

The subject of Case 7 presented with infiltrative erythema, edema anderythema on the face and neck, and papules and erythema weredisseminated on the four limbs. The rash on the face and neck was mainlycharacterized by severe infiltration, erythema and swelling. Theseverity level based on the “Dermatological Association Guideline” wassevere, and symptom evaluation based on the “Guideline 2005” was severe.Steroid external therapy was applied to this subject, but the subjecthad recurrence soon after its discontinuation.

Treatment and its Outcome:

The ANP gel-base preparation with a concentration of 50 μg/g obtained inExample 18 was applied to the subject of Case 7, but both erythema anditching worsened by drying. Therefore, the BNP gel-base preparation witha concentration of 30 μg/g obtained in Example 13 was applied to theface twice a day; after 2 to 3 days, erythema, edema, dryness, anditching were markedly improved. Itching sensation level was improvedfrom 10 prior to the application to 0.

BNP test example 8 (Case 8; subject 43):

The subject of Case 8 presented with infiltrative erythema withlichenification, erythema, many papules, and excoriations on the body,and infiltrative erythema, erythema and many papules on the face; therash on the face was mainly characterized by infiltrative erythema, manypapules, scales and excoriations. The severity level based on the“Dermatological Association Guideline” was severe, and symptomevaluation based on the “Guideline 2005” was most severe. Steroidexternal therapy was applied to this subject, but the subject hadrecurrence soon after its discontinuation.

Treatment and its Outcome:

The BNP aqueous-solution preparation with a concentration of 50 μg/mlobtained in Example 14 was applied to the subject of Case 8 twice a day;after 2 days, erythema, infiltration, scales, and itching were markedlyimproved. Itching sensation level was improved from 10 prior to theapplication to 3. FIG. 15 shows photographs before and after 5 days ofapplication. A shows the state before application, and B shows the stateafter application.

BNP test example 9 (Case 9; subject 44):

The subject of Case 9 presented with infiltrative erythema, erythema,edema, scales and numerous excoriations on the four limbs, body trunk,neck and face, and the rash on the face consisted of infiltration,erythema, severe scales and crusts. The severity level based on the“Dermatological Association Guideline” was severe, and symptomevaluation based on the “Guideline 2005” was severe. Steroid externaltherapy was applied to this subject, but the subject had recurrence soonafter its discontinuation.

Treatment and its Outcome:

The ANP gel-base preparation with a concentration of 50 μg/g obtained inExample 18 was applied to the subject of Case 9; while redness of theskin slightly disappeared after approximately 3 days, erythema did notfade away any more even after 7 days of further application, and scalesalso remained. Therefore, the BNP aqueous-solution preparation with aconcentration of 50 μg/ml obtained in Example 14 was applied to the faceand neck twice a day; then erythema, itching, and dryness were markedlyimproved on the next day. Itching sensation level was improved from 10prior to the application to 0.

BNP test example 10 (Case 10; subject 45):

The subject of Case 10 presented with skin flush with chills, edema, andinfiltrative erythema on almost the whole body, and the rash on theface, neck and four limbs consisted of severe swelling, skin flush, andedema. The severity level based on the “Dermatological AssociationGuideline” was severe, and symptom evaluation based on the “Guideline2005” was most severe. Due to side effects caused by a long period ofsteroid external therapy and its excessive application, the subject wasin a state of erythroderma.

Treatment and its Outcome:

The subject of Case 10 is the subject of Comparative case 3 in thecomparative test example in which the later-described ANP gel-basepreparation has been applied. The BNP aqueous-solution preparation witha concentration of 50 μg/ml obtained in Example 14 was applied to theface and neck of this subject twice a day; swelling, erythema, itching,and the sensation of tight-stretched skin were fairly improved the nextday, and 3 days later, infiltrative erythema, swelling, excoriations,and itching were markedly improved, but scales and dryness stillremained. Itching sensation level was improved from 10 prior to theapplication to 1.

Subsequently, the ANP aqueous solution with a concentration of 50 μg/gwas applied twice a day, then the areas of erythema extended in one day,and itching worsened to a level that could be clearly perceivedsubjectively.

Summary of Therapeutic Effects of BNP Gel-Base Preparation And BNPAqueous-Solution Preparation:

The above case studies clarified the following.

By administering skin external preparations comprising BNP, erythemawith severe swelling/edema/infiltration, erythema with lichenification,papules, and scales were markedly improved, or they were improved to themild symptoms mainly characterized by dryness, mild erythema, scales,etc., or to the minor rash mainly characterized by dryness with lessinflammation. In particular, by administering skin external preparationscomprising BNP, skin flush, infiltration, scales lichenification andburning sensation on the face of adult patients, which are hardlycurable and can disrupt the patients′ social lives, could bedramatically improved to a condition without any irritation symptoms.Any of these skin external preparations comprising BNP such as BNPaqueous-solution preparation and BNP gel-base preparation demonstratedalmost identical therapeutic effects.

Due to such effects of the skin external preparations comprising BNP,continuation of application for 3 to 4 days apparently reduced erythemaand infiltration, resulting in almost normal skin with fine texture insome cases. This could be a great relief for patients with severe atopicdermatitis.

In addition, by administering skin external preparations comprising BNP,improvement of vasodilation and inflammatory erythema was confirmed fromfindings in the upper dermis of the skin by dermoscopy. Furthermore, byadministering skin external preparations comprising BNP, it wasconfirmed that the texture of the skin surface becomes finer, scalesdecrease, and the skin becomes soft to the touch. These findings werealso confirmed by the findings in the upper dermis of the skin bydermoscopy. The fact that “the texture of the skin surface becomesfiner, scales decrease, and the skin become soft to the touch” isimportant in compensating for skin dryness and deterioration of barrierfunctions as well as preventing recurrence of inflammation. Such effectswere also observed in psoriasis; disappearance of scales and improvementof infiltration were confirmed. Neither local irritation symptom norsystemic side effect was observed at all by the BNP application. Inrosacea as well, improvement in capillary dilation and erythema wasobserved by the BNP application.

These effects of BNP therapeutic preparations for dermatitis were almostsimilar to those of CNP claimed in the priority application, whichinclude the following: subjective burning sensation and sensation ofheaviness were improved at approximately 20 min after the application,and at approximately 40 min after the application, improvement oferythema, infiltration and swelling were objectively observed. Moreover,after 2 to 3 days of application, erythema with severeinfiltration/edema/swelling, erythema with lichenification, papules andscales were markedly improved to symptoms mainly characterized bydryness, mild to minor erythema and scales, etc. The continuation of theeffects was also observed, and good skin conditions were maintained for5 days to around 2 weeks after discontinuation of the application.

Thereafter, even when erythema relapsed, it did not worsen as wasobserved before the application, and symptoms were merely mild, and itwas confirmed that stable conditions could be maintained. This deservesa special note since these effects could not have been obtained byconventional therapy, i.e., steroid external application. In addition,even when symptoms relapsed, it was confirmed that re-application to therash could lead to mild or minor rash by a smaller number of applicationthan the initial application.

Regarding the degree of improvement of the rash, the severity level ofthe rash based on the “Dermatological Association Guideline” wereimproved from severe to mild or minor. According to the severity levelof local symptoms based on the globally used “SCORAD index: ClinicalEvaluation”, severity levels for most of the items “Erythema”,“Edema/population”, “Oozing/crusting”, “Excoriation” and“Lichenification” were improved from stage 3 to stage 1, i.e., the mostmild state.

In the first place, the goal of treatment of atopic dermatitis is toachieve the following conditions in patients.

(1) No symptoms; if any, minor symptoms without any problem in dailylife, with a requirement of a low degree of drug therapy.

(2) While minor or mild symptoms persist, there is low possibility ofacute worsening; even when the symptoms worsen, they will not persistfor a long time.

The above case studies clearly confirmed that the skin externalpreparations comprising BNP of the present invention are able to achievethese conditions in patients.

EXAMPLE 18

Comparative test

1. Production of ANP Gel-Base Preparation:

For comparative testing, ANP gel-base preparations were prepared asfollows.

0.1 g of methyl parahydroxybenzoate (product name: Mekkins M, Ueno FineChemicals Industry), 0.2 g of phenoxyethanol, and 3.0 g of1,2-pentanediol were measured in the same container, dissolved at 60-70°C., and introduced into a mixing kettle. 6.0 g of concentrated glycerinwas introduced and a mixture of 0.44 g of carboxy vinyl polymer (productname: Carbopol 940, Lubrisol Advanced Materials, Inc.) and 0.08 g ofxanthan gum (product name: Keltrol T, CP Kelco) was added to thissolution and stirred thoroughly with a paddle at 15 rpm for dispersion.Then, while stirring with a paddle at 15 rpm, 83.95 g of purified waterwas gradually introduced, and the mixture was dissolved by stirring at akettle temperature of 70-80° C. using a paddle at 20 rpm and a disperserat 1500-2000 rpm. After stopping the disperser, dissolution wasconfirmed and cooling was immediately started; when the kettletemperature approached around 40° C., 6.0 g of Lubrajel NP from ISPJapan, Ltd. (2.7 g of glycerin, 0.06 g of carboxy vinyl polymer, 0.018 gof sodium polyacrylate, 3.222 g of water) was added and mixedhomogeneously with a paddle at 20 rpm, then 0.230 g of potassiumhydroxide was added for neutralization, and when the kettle temperaturereached 25° C., the rotation of the paddle was terminated to obtain agel base.

Then, 1000 μg of HANP injection 1000 (carperitide for injection;α-atrial natriuretic peptide preparation) as a principal agent wasdissolved in 10 ml of an injection solvent to obtain the ANP solutionwith a concentration of 100 μg/ml, and 10 ml of this ANP solution wasdiluted with 10 g of the gel-base obtained as above, to produce agel-base preparation with an ANP concentration of 50 μg/g.

In addition, in order to conduct the comparison under the same conditionas BNP, human ANP-28 (Peptide Institute, Inc.) was also used for theexamination. In this case, 0.5 mg of human ANP-28 was dissolved in 1 mlof purified water to obtain an ANP solution with a concentration of 500μg/ml, then 1.0 ml of this solution was homogeneously mixed and stirredin 9 g of the above gel-base to obtain the ANP gel-base preparation. TheANP concentration of this gel-base preparation is 50 μg/g.

EXAMPLE 19

2. Production of ANP Aqueous-Solution Preparation:

0.5 mg of human ANP-28 (Peptide Institute, Inc.) as a principal agentwas dissolved in 1 ml of purified water to obtain an ANP solution, and1.0 ml of this solution was diluted with 9 ml of purified water toproduce the aqueous-solution preparation with an ANP concentration of 50μg/ml.

EXAMPLE 20

3. Diagnosis of Subjects:

Patients′ diagnosis, evaluation of symptoms, selection of externaltherapy, test method and observation of the skin were performedsimilarly to the methods of Example 1.

In addition, similarly to the methods of Example 1, prior toadministration of the ANP gel-base preparation of the present invention,history taking from subjects, scratch tests for allergens, and diagnosiswere conducted. Table 21 (subjects 45-49) shows the results of thesubjects' history taking and diagnosis, i.e., sex, age, onset and courseof disease, family history, past history, scratch test result,diagnostic findings, and symptom evaluation based on the “Guideline2005” of the subject in each case.

EXAMPLE 21

4. Therapeutic Effects on Subjects:

Therapeutic effects of the ANP gel-base preparation of the presentinvention are shown in Table 22 (subjects 45-49). In Table 22, “itchingsensation” represents comparison of the itching sensation evaluatedusing visual analogue scale method before and after treatment.Similarly, “non-recurrence period” refers to the period afterdiscontinuation of the treatment by the preparation of the presentinvention subsequent to improvement of symptoms, for which relapse ofthe symptoms did not occur. In order to evaluate the resultsobjectively, photographs before and after the application of ANPpreparations were taken for all cases. Of these, photographs of some ofthe cases are shown in the figures.

TABLE 21 Diagnosis prior to application of ANP gel-base preparation.Subject Subject 45 Subject 46 Subject 48 (FIG. 17) (FIG. 16) Subject 47(FIG. 18) Subject 49 Case Case 3 Case 2 Case 4 Case 5 Case 1 Sex FemaleFemale Female Male Male Age 48 years old 27 years old 21 years old 28years old 14 years old Onset and Developed at 3 Developed in Developedin Developed in Developed in course of months of age; infancy; symptomsinfancy; symptoms infancy; symptoms infancy; symptoms disease symptomsworsen worsened since worsen in dry were mainly on worsened since asseasons the start of seasons, and the body trunk around the 2nd change.Recently menstruation in erythema with and four limbs; grade of the rashworsened the junior high itching appeared they extended to elementarydue to school, and they mainly on the the face and neck school, and heleisureless life, were alleviated face, neck, upper after he reachedvisited many and she is in a during pregnancy, limbs and trunk;adulthood. In clinics but they sate of but relapsed they relapse andparticular, the were not erythroderma. after birth, worsen due to rashon the back improved; extending to the steroids. shows intractablestarting 5 years whole body. nature. ago, dryness has becomeparticularly severe. Family Elder brother; Younger brother; Mother;Atopic Mother; Allergic history Atopic Bronchial asthma, dermatitisrhinitis, dermatitis, Mother; Allergic Allergic rhinitis conjunctivitisPast Allergic rhinitis Allergic rhinitis Allergic Allergic rhinitisBronchial asthma, history rhinitis, Allergic Allergic rhinitis,conjunctivitis Allergic conjunctivitis Scratch House dust: 3+ Housedust: 2+ House dust: 1+ House dust: 1+ House dust: 3 + test Mite: 3+Mite: 3+ Mite: 1+ Mite: 2+ Mite: 3+ Cedar: 2+ Cedar: 2+ Cedar: 1+ Cedar:3+ Orchard grass: 3+ Orchard grass: 2+ Orchard grass: 3+ Orchard grass:2+ Ragweed: 1+ Diagnostic Skin flush with Infiltrative InfiltrativeInfiltrative Erythema with findings chills, edema, erythema witherythema, erythema, strong itching and infiltrative lichenification,erythema, severe erythema, associated with erythema are erythema, andscales, adhesion numerous sleep observed on the severe scales are ofcrusts, and excoriations, and disturbance, almost whole observed on thenumerous papules are severe scales, body. face and neck, excoriationsare particularly and numerous infiltrated observed over the severelyobserved excoriations are erythema is whole body, and on the back.observed over the observed on the they are whole body. four limbs andparticularly body trunk. severe on the face and neck. Symptoms Rash onthe face, Rash on the face Rash on the face Rash on the back Rash on theback of neck, and four is mainly is mainly consists of is mainlyapplication limbs consist of characterized by characterized by severecharacterized by regions severe swelling, severe severe infiltration,severe scales, skin flush and lichenified infiltration, erythema,erythema, and edema. infiltration, edema, erythema, numerous numerouserythema, and scales, crusts excoriations, excoriations. scales. andexcoriations. papules and lichenification. Effects She is in a stateSteroid external Symptoms on the Sufficient Symptoms are of oferythroderma therapy is not face soon relapse effects cannot betemporarily steroid due to the side sufficiently with the use ofobtained with reduced with external effects by long- effective tosteroid, and steroid external steroid external drug term steroidinfiltrative further worsen application, and therapy, but soon externaltherapy erythema on the compared to the symptoms soon relapse and andexcessive face. condition before relapse strong dryness occurs.application. the use. itching does not subside. Evaluation Most severeMost severe Severe Severe Most severe of symptoms

TABLE 22 Therapeutic effects of ANP gel-base preparation. SubjectSubject 45 Subject 46 Subject 48 (FIG. 17) (FIG. 16) Subject 47 (FIG.18) Subject 49 Case Case 3 Case 2 Case 4 Case 5 Case 1 Sex Female FemaleFemale Male Male Age 48 years old 27 years old 21 years old 28 years old14 years old Dosage form ANP gel-base ANP gel-base ANP gel-base ANPgel-base ANP gel-base preparation preparation preparation preparationpreparation Dosage 50 μg/g 50 μg/g 50 μg/g 50 μg/g 50 μg/g Number ofTwice a day Twice a day Twice a day Twice a day Twice a dayadministration Days of 7 days 7 days 5 days 5 days 7 days administrationApplied region Face and upper Face and neck Face Back Back limbsSeverity level Before: severe Before: severe Before: severe Before:severe Before: severe of rash by After: severe After: severe After:severe After: severe After: severe Dermatological Association GuidelineSeverity level Erythema: 3 Erythema: 2 Erythema: 3 Erythema: 3 Erythema:2 evaluation of Edema/papulation: 3 Edema/papulation: 2Edema/papulation: 2 Edema/papulation: 2 Edema/papulation: 1 the rashOozing/crusting: 3 Oozing/crusting: 1 Oozing/crusting: 3Oozing/crusting: 3 Oozing/crusting: 2 region by Excoriation: 1Excoriation: 1 Excoriation: 2 Excoriation: 3 Excoriation: 2 SCORAD(before Lichenification: 2 Lichenification: 2 Lichenification: 2Lichenification: 3 Lichenification: 2 application) Dryness: 3 Dryness: 3Dryness: 2 Dryness: 2 Dryness: 3 Total: 15/18 Total: 11/18 Total: 14/18Total: 16/18 Total: 12/18 Severity level Erythema: 3 Erythema: 3Erythema: 3 Erythema: 3 Erythema: 2 evaluation of Edema/papulation: 2Edema/papulation: 2 Edema/papulation: 2 Edema/papulation: 3Edema/papulation: 1 the rash Oozing/crusting: 3 Oozing/crusting: 2Oozing/crusting: 2 Oozing/crusting: 3 Oozing/crusting: 2 region byExcoriation: 2 Excoriation: 1 Excoriation: 2 Excoriation: 3 Excoriation:2 SCORAD (after Lichenification: 2 Lichenification: 2 Lichenification: 2Lichenification: 3 Lichenification: 2 application) Dryness: 3 Dryness: 3Dryness: 2 Dryness: 2 Dryness: 3 Total: 15/18 Total: 13/18 Total: 13/18Total: 17/18 Total: 12/18 Detailed By the Despite the Despite theDespite the Despite the description of application of 50 μg/gapplication of 50 μg/g application of 50 μg/g application of 50 μg/gapplication of 50 μg/g improvement ANP gel-base ANP gel-base ANPgel-base ANP gel-base ANP gel-base status of preparation twicepreparation twice preparation twice preparation twice preparation twicesymptoms a day, edema was a day for 7 days, a day, erythema a day for 3days, a day for 7 days, slightly reduced, redness did not andinfiltration erythema and severe scales, but skin flush disappear andwere not reduced itching were not erythema and and erythema wereerythema and at all even after improved and numerous not improved evenscales worsened. 5 days. edema even excoriations after 7 days. worsened.still remained just as the condition before application. Itching Before:10 Before: 10 Before: 10 Before: 10 Before: 10 sensation After: 10After: 10 After: 10 After: 10 After: 10 Non-recurrence — — — — — period

EXAMPLE 22

For comparison, a test was performed using the ANP gel-base preparationwith a concentration of 50 μg/g obtained in Example 18. For reference,as photographs before and after application, FIG. 16 shows photographsof Comparative test example 2 (Case 2; subject 46), FIG. 17 showsphotographs of Comparative test example 3 (Case 3; subject 45), and FIG.18 shows photographs of Comparative test example 5 (Case 5; subject 48).

Comparative test example 1 (Case 1; subject 49):

The subject of Comparative case 1 presented with erythema with strongitching associated with sleep disturbance, severe scales and numerousexcoriations over the whole body. In addition, the rash on the back wasmainly characterized by severe scales, erythema and numerousexcoriations, and the severity level based on the “DermatologicalAssociation Guideline” was severe. The symptom evaluation of thissubject based on the “Guideline 2005” was most severe, and the symptomswere reduced temporarily by steroid external therapy, but they relapsedimmediately and the skin dried.

Treatment and its Outcome:

The ANP gel-base preparation with a concentration of 50 μg/g obtained inExample 18 was applied to the back of the subject of Comparative case 1twice a day for 7 days, but the severe scales, erythema and numerousexcoriations remained unchanged compared to the condition before theapplication. The itching sensation level before application was 10, andit was still 10 after application; the severity level remained severeafter application.

Comparative test example 2 (Case 2; subject 46):

The subject of Comparative case 2 presented with infiltrative erythemawith lichenification, erythema, and severe scales on the face and neck,and infiltrative erythema on the four limbs and body trunk. The rash onthe face was mainly characterized by severe lichenified infiltration,erythema and scales, and the severity level based on the “DermatologicalAssociation Guideline” was severe. The symptom evaluation of thissubject based on the “Guideline 2005” was most severe, and sufficienteffects of steroid external therapy were not observed for theinfiltrative erythema on the face.

Treatment and its Outcome:

The ANP gel-base preparation with a concentration of 50 μg/g obtained inExample 18 was applied to the face and neck of the subject ofComparative case 2 twice a day for 7 days, but redness did notdisappear, and both erythema and scales worsened. FIG. 16 showsphotographs before and after 5 days of application. A shows the statebefore application, and B shows the state after application. The itchingsensation level before application was 10, and it was still 10 afterapplication; the severity level remained severe after application.

Comparative test example 3 (Case 3; subject 45):

The subject of Comparative case 3 presented with skin flush with chills,edema, and infiltrative erythema on the almost whole body. The rash onthe face, neck, and four limbs consists of severe swelling, skin flush,and edema, and the severity level based on the “DermatologicalAssociation Guideline” was severe. The symptom evaluation of thissubject based on the “Guideline 2005” was most severe, and due to theside effects caused by a long period of steroid external therapy and itsexcessive application, the subject was in a state of erythroderma.

Treatment and its Outcome:

The ANP gel-base preparation with a concentration of 50 μg/g obtained inExample 18 was applied to the face and upper limbs of the subject ofComparative case 3 twice a day, then edema was slightly reduced, butboth skin flush and erythema was not improved even after 7 days ofapplication. FIG. 17 shows photographs before and after days ofapplication. A shows the state before application, and B shows the stateafter application. The itching sensation level before application was10, and it was still 10 after application; the severity level remainedsevere after application.

Comparative test example 4 (Case 4; subject 47):

The subject of Comparative case 4 presented with infiltrative erythema,erythema, severe scales, adhesion of crusts, and numerous excoriationsover the whole body, and they are particularly severe on the face andneck. The rash on the face was mainly characterized by severeinfiltration, edema, erythema, papules, scales, crusts and excoriations,and the severity level based on the “Dermatological AssociationGuideline” was severe. The symptom evaluation of this subject based onthe “Guideline 2005” was severe; and despite the use of steroids,symptoms on the face soon relapsed and they tended to worsen compared tothose before the application.

Treatment and its Outcome:

The ANP gel-base preparation with a concentration of 50 μg/g obtained inExample 18 was applied to the face of the subject of Comparative case 4twice a day, but both erythema and infiltration was not reduced evenafter 5 days of application. The itching sensation level beforeapplication was 10, and it was still approximately 10 after application;the severity level remained severe after application.

Comparative test example 5 (Case 5; subject 48):

The subject of Comparative case 5 presented with infiltrative erythema,erythema, numerous excoriations and papules particularly on the back.The rash on the back consists of severe infiltration, erythema, numerousexcoriations, papules and lichenification, and the severity level basedon the “Dermatological Association Guideline” was severe. The symptomevaluation of this subject based on the “Guideline 2005” was severe, andsufficient effects were not observed by steroid external application,the symptoms relapsed soon, and strong itching was not reduced.

Treatment and its Outcome:

The ANP gel-base preparation with a concentration of 50 μg/g obtained inExample 18 was applied to the back of the subject of Comparative case 5twice a day, but no effect was observed for both erythema and itchingafter 3 days of application. FIG. 18 shows photographs before and after5 days of application. A shows the state before application, and B showsthe state after application. The itching sensation level beforeapplication was 10, and it was still 10 after application; the severitylevel remained severe after application.

Summary of the comparative test examples:

The above comparative case studies clarified the following.

According to the comparative tests using external preparationscomprising ANP, the ANP external preparations with the sameconcentration as CNP or BNP exhibited marginal effects in a certaincases; however, onset of the effects required approximately 3 days ofapplication, and erythema did not disappear completely; even whenapplication was continued for 7 days or more, erythema did not disappearas in the cases of therapeutic agents for dermatitis comprising CNP orBNP, and erythema even worsened in some cases. Furthermore, when theexternal application was discontinued, symptoms soon relapsed anddryness increased, with enhanced itching. Apparently, these effects wereinferior to those of the therapeutic agents for dermatitis comprisingCNP or BNP. In many cases, ANP external preparations did not improve theconditions or even worsened the conditions, and improvement of the rashwas, in terms of the severity level of the rash by the “DermatologicalAssociation Guideline”, from severe to severe, remaining at the samelevel. According to the severity level of local symptoms based on theglobally used “SCORAD index: Clinical Evaluation” including the items“Erythema”, “Edema/papulation”, “Oozing/crusting”, “Excoriation” and“Lichenification”, severity levels for “Edema/papulation” and“Oozing/crusting” were improved from stage 3 to stage 2 in some of thecases; however, those for “Excoriation,” “Lichenification” and“Erythema” remained at the same level of stage 3.

In the first place, the goal of treatment of atopic dermatitis is toachieve the following conditions in patients.

-   (1) No symptoms; if any, minor symptoms without any problem in daily    life, with a requirement of a low degree of drug therapy.-   (2) While minor or mild symptoms persist, there is low possibility    of acute worsening; even when the symptoms worsen, they will not    persist for a long time.

The above comparative case studies confirmed that the skin externalpreparation comprising ANP was unable to achieve these conditions inpatients.

Comprehensive summary of the effects of the invention

In order to confirm the effects of the skin external-preparationcomposition of the present invention in overview, composite indices madeby severity evaluation of rash regions based on SCORAD for eachpreparation were compared between before and after application, and theresult of the comparison was expressed with a bar graph as shown in FIG.19.

Severity levels of rash regions based on SCORAD compared between beforeand after application of the ANP gel-base preparations as comparativetest examples showed almost no difference. In contrast, when the skinexternal-preparation compositions comprising CNP or BNP were used, theseverity level of rash regions based on SCORAD was dramatically improvedafter application.

Similarly, in order to confirm the effects of the skinexternal-preparation compositions of the present invention in apanoramic manner, itching sensation evaluated using visual analoguescale method for each preparation were compared between before and afterapplication, and the result of the comparison was expressed with a bargraph, as shown in FIG. 20.

There was no change in the itching sensation before and afterapplication of the ANP gel-base preparations as comparative testexamples. In contrast, when the skin external-preparation compositionscomprising CNP or BNP were used, the itching sensation level wasdramatically improved after application.

Thus, the results shown in FIGS. 19 and 20 demonstrated that the skinexternal-preparation compositions comprising CNP or BNP of the presentinvention dramatically improved symptoms of atopic dermatitis.

INDUSTRIAL APPLICABILITY

The skin external-preparation compositions comprising CNP or BNP, inparticular the therapeutic preparations for dermatitis, of the presentinvention, are extremely effective in the treatment of atopicdermatitis, which is recognized to have particularly-intractable nature.And the preparations of the present invention are capable of wideapplication. In addition, there is little concern of systemic sideeffects since CNP and BNP are originally intrinsic hormones, since thenumber of days of external application is only approximately 3 days,since an amount taken into the body through absorption from the skinupon being used as a topical medication is extremely small, and since aconcentration used in the present invention is merely around 30 μg/g.The preparations of the present invention dramatically reduce subjectiveitching sensation without causing local irritation symptoms. Hence, thepreparations of the present invention can be applied to patients in whomconventional steroids and tacrolimus were ineffective, as well as topatients and young subjects in whom steroids and tacrolimus cannot beused due to the concern of side effects. Moreover, the QOL of psoriasispatients who suffer from many scales can be improved.

Therefore, research and development as well as practical application ofthe present invention as novel therapeutic preparations for dermatitiscan be greatly expected.

Furthermore, the preparations of the present invention have an efficacyas skin-care cosmetics that improve texture of the skin, since theeffects with the preparations of the present invention that “the textureof the skin surface becomes finer, scales decrease, and the skin becomesoft to the touch” is important in compensating for skin dryness and thedeterioration of barrier functions as well as preventing recurrence ofinflammation, and since the preparations of the present invention exertthe effects regardless of dosage form.

Namely, effects of anti-inflammation, horny cell layer care, epidermiscare, and basal membrane care are observed, and in terms of clinicaleffects, improvement of elasticity and wrinkles of the skin, andmoisturizing the skin are observed. From such a point of view, BNPtherapeutic preparations for dermatitis of the present invention arealso expected for their practical application as a skin-textureimproving agent.

What is claimed is:
 1. A method for treating dermatitis, comprising:externally applying a composition comprising an effective amount of anatriuretic peptide of C-type natriuretic peptide (CNP) or B-typenatriuretic peptide (BNP) to skin of a subject in need of suchtreatment, wherein: said CNP comprises the human amino acid sequence ofCNP-22 or CNP53, and said BNP comprises the human amino acid sequence ofBNP-26, BNP-32, or BNP-45.
 2. The method for treating dermatitisaccording to claim 1, wherein the natriuretic peptide is CNP-22.
 3. Themethod for treating dermatitis according to claim 1, wherein thenatriuretic peptide is BNP-32.
 4. The method for treating dermatitisaccording to claim 1, wherein the composition comprises 1-500 μg/g ofsaid C-type natriuretic peptide (CNP) or said B-type natriuretic peptide(BNP) based on the weight of the composition.
 5. The method for treatingdermatitis according to claim 1, wherein the composition comprises20-200 μg/g of said C-type natriuretic peptide (CNP) or said B-typenatriuretic peptide (BNP) based on the weight of the composition.
 6. Themethod for treating dermatitis according to claim 1, wherein thecomposition comprises 30-100 μg/g of said C-type natriuretic peptide(CNP) or said B-type natriuretic peptide (BNP) based on the weight ofthe composition.
 7. The method for treating dermatitis according toclaim 1, wherein the dermatitis is selected from the group consisting ofatopic dermatitis, dermatitis that led up to steroid dermatitis, steroidresistant dermatitis, dermatitis to which tacrolimus is not applicable,chronic dermatitis, erythroderma, eczema, contact dermatitis, seborrheicdermatitis, autosensitization dermatitis, stasis dermatitis, urticaria,drug eruption, dermal vasculitis, prurigo, pruritus cutaneus erythema,psoriasis, rosacea, rosacea like dermatitis, lichen planus, andfollicular keratosis.
 8. The method for treating dermatitis according toclaim 1, wherein the dermatitis is an inflammation associated with atleast one rash symptom selected from the group consisting of erythema,infiltrative erythema, lichenified lesion, scales, adhesion of crusts,eczema, abrasion, excoriation, prurigo nodularis, papule, erosions,infiltration, vesicle, and edema.
 9. The method for treating dermatitisaccording to claim 1, wherein the dermatitis shows an immune reaction toat least one allergen selected from the group consisting of house dust,mites, cedar pollen, orchard grass pollen, ragweed pollen, egg white,and egg yolk.
 10. The method for treating dermatitis according to claim1, wherein the dermatitis occurs in at least one region selected fromthe group consisting of face, neck, back, and arms.
 11. The method fortreating deiinatitis according to claim 1, wherein the dosage form ofthe composition is selected from the group consisting of ointment, gel,cream, lotion, solution, spray, and patch.
 12. A method for improvingskin texture, comprising: externally applying a composition comprisingan effective amount of a natriuretic peptide of C-type natriureticpeptide (CNP) or B-type natriuretic peptide (BNP) to skin of a subjectin need of such treatment, wherein: said CNP comprises the human aminoacid sequence of CNP-22 or CNP-53; and said BNP comprises the humanamino acid sequence of BNP-26, BNP-32, or BNP-45.
 13. The method forimproving skin texture according to claim 12, wherein the natriureticpeptide is CNP-22.
 14. The method for improving skin texture accordingto claim 12, wherein the natriuretic peptide is BNP-32.
 15. The methodfor improving skin texture according to claim 12, comprising 1-500 μg/gof C-type natriuretic peptide (CNP) or B-type natriuretic peptide (BNP)based on the weight of the composition.
 16. The method for improvingskin texture according to claim 12, comprising 20-200 μg/g of C-typenatriuretic peptide (CNP) or B-type natriuretic peptide (BNP) based onthe weight of the composition.
 17. The method for improving skin textureaccording to claim 12, comprising 30-100 μg/g of C-type natriureticpeptide (CNP) or B-type natriuretic peptide (BNP) based on the weight ofthe composition.
 18. The method for improving skin texture according toclaim 12, wherein improving skin texture is improving dry skin, roughskin, sensitive skin or fine wrinkles.
 19. The method for improving skintexture according to claim 12, wherein the composition is a skin careproduct or a quasi drug.
 20. The method for improving skin textureaccording to claim 12, wherein the dosage form of the composition iscream, foam, skin lotion, facial mask, skin-softening water, skinemulsion, foundation, makeup base, essence, soap, liquid cleanser, bathagent, sun-block cream, suntan oil or spray type liquid preparation.